i

Seasonal variations in some physiological responses of maize (Zea mays L.) under glasshouse conditions

By

Iqbal Hussain M.Sc. Botany (BZU)

Regd. No. 2004-ag-504

A thesis submitted in partial fulfillment of the requirements for the Degree of

DOCTOR OF PHILOSOPHY

IN

BOTANY

FACULTY OF SCIENCES

UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN

2009

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DECLARATION

I hereby declare that the contents of the thesis, entitled “Seasonal variations in some

physiological responses of maize (Zea mays L.) under glasshouse conditions” are product of

my own research and no part has been copied from any published source (except the

references, standard mathematical or genetic models/equation/formulae/protocols etc.). I

further declare that this work has not been submitted for award of any other

diploma/degree. The University may take action if the information provided is found

inaccurate at any stage.

Iqbal Hussain Regd. No. 2004-ag-504

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The Controller of Examinations University of Agriculture Faisalabad

We, the Supervisory Committee, certify that the contents and form of thesis

submitted by Iqbal Hussain, 2004-ag-504 have been found satisfactory and

recommend that it be processed for evaluation by the External Examiners for the

award of degree.

SUPERVISORY COMMITTEE 1. Chairman ____________________________ Prof. Dr. Abdul Wahid 2. Member ___________________________ Prof. Dr. Muhammad Ashraf 3. Member ___________________________ Prof. Dr. Shahzad M.A. Basra

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Acknowledgement 

I would like to express many sincere thanks to my supervisor Dr. Abdul Wahid Professor, Department of Botany, University of Agriculture, Faisalabad, who provided me encouragement, enthusiasm, guidance and support during my Ph.D research work, which enabled the production of this thesis and also helping me to overcome the difficulties of written language while writing this thesis

I also would like to acknowledge members supervisory committee supervisors, Dr. Muhammad Ashraf, Professor, Department of Botany and Dr. Shahzad M.A. Basra, Professor, Department of Crop Physiology, University of Agriculture, Faisalabad for reminding me of Physiological knowledge, their availability when needed, endless discussions during the research work

I also would like to acknowledge great cooperation rendered by Dr. Tadasi Sato and Dr. Atsushi Higashitani, Graduate School of life Sciences, Tohoku University, Sendai, Japan during my visit to their lab under IRSIP program of HEC, Islamabad, Pakistan. They brought me to a new fabulous area of molecular biology with modern quantitative RT-PCR techniques. Many thanks also go to Dr. Tadashi Sakata, Dr. Kazahiro Sasaki, Eiko Hanzawa, Yuri Sann, Lee Hyun Sook, Kuriyama Satohiro, Chihiro Mori, Tomabechi, Shishiki Sann, Takeshi Oshino, Miura Shinya and Takafami Kimura for their kind assistance while conducting molecular biology experiments and also helping me with preliminary inception of RT-PCR experiment

I would extend my profound gratitude to Dr. Muhammad Yasin Ashraf, NIAB Faisalabad for providing facilities for physiological studies. Thanks also go to Dr. Furrakh Javed, Assistant Professor, Department of Botany for allowing the use of spectrophotometer. My Thanks are due to those people listed hereafter who

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have assisted me during difficult hours of work while I was conducting my Ph.D research: Dr. Sadia Gelani, Dr. Freeha Anjum, Mrs. Saqib Mahmood and Mr. Rizwan Rasheed. Many thanks are extended to Mr. Muhammad Saleem Anjum and Mr. Abid Mustafa for helping me setup glass canopies and helping with purchasing chemicals and equipment.

I am also grateful to all my friends especially Mr. Ejaz Hussain Siddiqi, Maqsood Iqbal Shami, Azam Zia, Abid Hussain, Zia Ullah, Aftab Allam, Irfan Azhar, Malik Munawar, Zafar Iqbal Javed, Munawer Ahmad Khan, Muhammad Arshad Dar, Mujahid Ahmad Qaiserani, Hasnat Abbas, Mukhtar Hussain, Zawar Hussain, Khadim Hussain, Abid Khan,Ejaz Ahmad,Ghulam Abbas Syed Seqlain Raza and Syed Muhammad Kazim Raza for their support and encouragement during the course of my studies and research.

My deep gratitude is given to the Government of Pakistan for having granted me doctoral scholarship under Indigenous 5000 fellowship program sponsored by Higher Education Commission, Pakistan (HEC) and my visit to Japan for six months training under International Research Support Initiative Program (IRSIP) because without this, it would not have been completed smoothly and rapidly.

I also pay gratitude to the anonymous external

examiners who greatly improved the thesis to bring it into a final shape.

I dedicate this work to my family for their

lifetime love and support. I owe my deepest gratitude to my parents, my dear mother Hayat BiBi and my father Muhammad Siddique, for nurturing me and inspiring to pursue doctorate education. Thanks are due to all my sisters and brother Altaf Hussain for their psychological encouragements. Finally, I wish to thanks my beloved wife Rubina Iqbal and children, Muhammad Muazzam Ali, Aemon Iqbal, Zoha Iqbal and Mahnoor Iqbal

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for their patience, continuously encouragement, inspiration and psychologicaly supported me to overcome the most desperate moments while completing this Ph.D progamme.

Iqbal Hussain

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TABLE OF CONTENTS 

S. NO. TITLE PAGE

I INTRODUCTION 01

II REVIEW OF LITERATURE 06

III MATERIALS AND METHODS 28

IV RESULTS AND DISCUSSION 41

V SUMMARY 109

LITERATURE CITED 111

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ABSTRACT

In view of the changing climatic conditions mainly related to greenhouse effect, this study was

focused on determining the responses of two differentially heat tolerant maize varieties to

glasshouse condition. The parameters studies included growth, water relations, gas exchange,

photosynthetic pigments, oxidative damage and gene expression. Results revealed that prevailing

glasshouse conditions played a crucial role in affecting the maize growth across winter and

summer seasons. Despite differences in the growing seasons and varieties glasshouse conditions

were adverse for the photosynthetic systems in maize. Major yardsticks of sensitivity were loss

of chlorophyll and carotenoids in the light reactions, while reductions in the net photosynthesis

and stomatal conductance in the glasshouse grown maize. Prevailing glasshouse conditions were

greatly effective in hampering the leaf water relation particularly those of winter sown crop. The

glasshouse conditions in winter crop produced oxidative stress on the plants, which was explicit

from the increased synthesis of H2O2, MDA and increased permeability to the ion leakage.

Greater free proline accumulation in the tolerant variety not only presented itself as a major

amino acid accumulated in environmental stress tolerance but also indicated it as a reliable

indicator of tolerance to glasshouse condition in maize. With great varietal difference, changes of

temperature and relative humidity inside the glasshouse across the seasons were mainly

responsible for the observed changes in mineral nutrients. More distinct changes were evident in

K, Ca and nitrate nutrition, which were given greater credence in view of their closer association

to the seasonal changes in the environmental conditions inside the glasshouse. Maize seedlings

showed sensitivity to high temperature stress, which was recorded from morphological

(reduction in shoot fresh weight, dry weight of shoot and root and a reduction in fresh-to-dry

weight ratio) and gene expression patterns. The molecular studies suggested that the maize

sensitivity to high temperature was mainly due to enhanced coexpression of sag and dhn2 and

failure to express hsp70 and sgr2 during relatively long term exposure to high temperature.

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CHAPTER-1 INTRODUCTION

Crop plants such as maize, sugarcane and sorghum are referred to as C4 plants. They have a

distinctive leaf anatomy and photosynthetic metabolism that concentrates carbon dioxide (CO2)

around rubisco as compared to rubisco of C3 plants such as cotton and soybean (Taiz and Zeiger,

2006). Plants with C4 anatomical and biochemical specialization show much less adverse effects

of elevated atmospheric CO2 (Ca) as compared to non-C4 plants. According to Ghannoum et al.

(2000), photosynthesis and growth of C4 plants positively respond to elevated Ca. The leaf

development and net photosynthesis in maize are maximum near 31oC (Tollenaar, 1989; Yan and

Hunt, 1999) and 34oC (Kim et al., 2007) at ambient Ca, which decreased at temperature above

37oC while complete inhibition took place near 45oC (Crafts-Brandner and Salvucci, 2002). High

temperature caused a reduction in shoot dry weight, relative growth rate and net assimilation rate

in maize (Ashraf and Hafeez, 2004). The rate of respiration, growth and dry matter yield varied

with temperature in maize (Tollenaar, 1989).

The plant growth, its development and leaf photosynthetic rate may not change at

elevated Ca in response to enrichment of CO2 in maize, but significantly change under high

temperature (Kim et al., 2007). Short-term studies by Drake et al. (1991) concluded that elevated

CO2 increase the net rate of photosynthesis and growth while long-term CO2 enrichment

decreases photosynthesis due to acclimation response in C4 plants (Kim et al., 2006; Leakey et

al., 2006). Recent climatic model predicts that ambient temperature around the globe may

increase 1.1 to 6.4oC with the doubling of atmospheric carbon dioxide (Kim et al., 2007). It is

believed that global climate is changing so exposure to high temperature is likely to increase.

According to Intergovernmental Panel on Climatic Change (IPCC), there was a 0.5oC increase

during the past 100 years and it is expected that our earth will be 0.2oC warmer per decade for

the next two decades and 1 to 3.4oC warmer in the year 2100 (IPCC, 2007), and this global

warming will have a great impact on agriculture.

High temperature that may interfere with pollen mother cells and microspores

development and causes male sterility in various plant species (Sakata et al., 2000; Abiko et al.,

2005). A gradual increase in temperature every year is likely to change cropping pattern, growth

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and economic yield of many important crops (Wollenweber et al., 2003). Gradually increasing

temperature is a great threat to agricultural production all over the world. Heat stress is a serious

problem to agricultural productivity all over the world, particularly in tropical areas. In plants

optimum temperature is essential for reproduction and maximum yield (Cheikh and Jones, 1994;

Keeling et al., 1994). Some reports show that an increase in temperature by a single degree

above normal can lead to a significant reduction in growth and yield (Pastori and Foyer, 2002).

High temperature affects morphological, biochemical and physiological processes in plants and

the major effects entail scorching of aerial plant parts, sunburn of branches and stems, leaf death,

leaf abscission and senescence, and causes inhibition of shoot and root growth, and reduced yield

(Ismail and Hall, 1999; Wahid et al., 2007). High temperature stress leads to premature

development of anther and restricts cell proliferation (Oshino et al., 2007).

Heat stress has a harsh effect on many economically important cereals plants such as

wheat, rice, maize etc., and decreases their yield by affecting the reproductive stages of these

plants. It also decreased chlorophyll content, net photosynthetic rate and stomatal conductance

(Morales et al., 2003). Photosynthesis is of pivotal importance for carbon accumulation,

production of biomass in different plant species. Response of terrestrial plants to photosynthesis

can changed ecosystem balance and cycling of carbon under global warming (Gunderson et al.,

2000). Increased ambient temperature affects plant productivity by damaging photosynthesis

(Al-Khatib and Paulsen, 1990). Photosynthetic inhibition can be reversible when temperature is

slightly supra-optimal (Berry and Bjorkman 1980). The yield of PSII reaction centers and

amount of RUBP inhibit net photosynthesis at high temperature (Law and Crafts-Brandner,

1999).

Sudden heat stress may injure the membranes by denaturing membrane proteins or

increase in unsaturated fatty acids, leading to membrane rupture and loss of cellular contents

(Savchenko et al., 2002). Heat stress induces deterioration of biological membranes, resulting in

ion leakage and deactivation of membrane proteins, and loss of cellular functions. Membrane

damage occurs mainly due to (1) stress induced production of activated oxygen species (AOS)

and reactive oxygen species (ROS) and (2) dehydration induced changes in phase transitions

(Nishida and Murata, 1996; Liu and Huang, 2000). Electrolyte leakage is measured to detect

stress injury to cell membranes in order to assess the severity of existing stress (Foyer et al.,

1997; Al-Khatib and Paulsen, 1999). Electrolyte leakage varies in relation to membrane abilities

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to take up and retain solutes and reflect stress induced changes in their potentials. Studies show

that search for genotypic variation in high temperature tolerance on the basis of leaf electrolyte

leakage may greatly influence the plants already exposed to this adversary (Li et al., 1991).

Membrane stability is positively associated with yield performance under heat stress (Rahman et

al., 2004). The AOS and ROS react with pigments, membranes, enzymes and nucleic acids, and

modify their functions (Smirnoff, 1993; Sairam et al., 2000). Heat stress decreases the activities

of antioxidant enzymes, leaf senescence and injury to cell membranes by increasing the level of

lipid peroxidation (Liu and Huang, 2000).

Heat stress has well marked effect on osmotic, turgor and water potential (Machado and

Paulsen 2001; Wahid and Close, 2007). Changes in plant processes like assimilate partitioning,

hampered water and nutritional relationships by the root has been observed under heat stress

(Morales et al., 2003; Ashraf and Hafeez, 2004; Wahid and Close, 2007). One of such

mechanisms is synthesis of specific proteins (Vierling, 1991) called heat shock proteins (HSPs).

All living organisms respond at the molecular level to thermal shock or other stresses by

inducing or enhancing the expression of a small number of specific heat shock protein (hsp)

genes and synthesis of their transcripts and showing the biosynthesis of stress proteins as a

stress-tolerance approach (Sun and Callis, 1997; Iba, 2002; Wahid and Close, 2007). Expression

of HSPs is an essential adaptive strategy for heat tolerance (Feder and Hoffman, 1999). The

HSPs, synthesized over a wide range size (10 kDa to 200 kDa) have chaperone-like functions

and are involved in signal transduction during heat stress (Schöffl et al., 1999). Rapid synthesis

of HSPs can be important for the protection of metabolic machinery of the cell (Camejo et al.,

2005; Wahid and Shabbir, 2005; Momcilovic and Ristic, 2007).

In Pakistan, maize (Zea mays L.) is the third most important cereal grain after wheat and

rice. Maize is essential for global food safety, since it is a multipurpose and commercial crop and

used as food, feed, fodder and agro-based industrial use (Dowswell et al., 1996; Herald et al.,

1996). It has high nutritive value and has been reported to contain about 72% starch, 10% protein

(Zein), 4.8% oil of good quality, 9.8% fiber, 3.0% sugar and 1.7% ashes (Choudhary, 1983;

Okoruwa, 1995). The oil of maize is of good quality. Maize is a short duration and fast growing

crop, having great adaptability to existing cropping systems and shows high yield potential. It is

an important summer crop in Pakistan and is grown on an area of 1.022 million hectares and its

total production is 3.560 million tons and an average grain yield of 3483 kg ha-1. Unfortunately,

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the grain yield of maize is very low as compared to other countries (Anonymous, 2008). This is

mainly due to the physical constraints that plants usually encounter under field conditions such

as environmental stresses especially heat stress that may interfere with the crop productivity.

Heat stress readily changes the pattern of gene expression, as a significant part of

thermotolerance (Yang et al., 2006). It also weakens the mRNAs encoding non-heat-stress-

induced proteins (Gallie et al., 1995). The organisms suddenly exposed to high temperature;

synthesize a small amount of heat shock proteins (HSPs). Increased expression of HSPs is

mediated at multiple levels, i.e., mRNA synthesis and its stability, and translation efficiency. The

heat shock response is conserved reaction of cells and high temperature. It is found that plant

reproductive development and molecular response to stresses required specific gene expression

(Sorensen et al., 2002; Shinozaki et al., 2003; Chinnusamy et al., 2004). Specific heat shock

protein genes, transcripts accumulation biosynthesis of stress proteins is altered under several

stresses (Ozturk et al., 2002).

Maize pollen development and tube growth are more sensitive to high temperature

(Gagliardi et al., 1995; Mascarenhas and Crone, 1996). The appearance of heat shock protein 70

(HSP70) and heat shock factor (hsf) gene is high at the beginning of maize pollen exposure to a

heat shock. This defect is not associated to an alteration of hsf genes (Zmhsf a-c) expression at

the mRNA level (Gagliardi et al., 1995). The studies show that under heat stress, distinctive hsps

are not synthesized in germinating pollen. The hsp18 and hsp70 genes are transcribed in

response to heat shock but low level of the mRNA accumulation.

Photosynthesis is of fundamental importance for growth and biomass accumulation in

different plant species. High temperature accelerates the senescence, result decrease the

assimilation rate in grains (Spano et al., 2003). Leaf senescence is controlled by genetic and

abiotic factors (Nooden and Leopold, 1978). In leaf senescence, expression of gene is a complex

process. A large number of senescence associated genes (sags) and defensive genes has been

reported during the leaf senescence (Lee et al., 2001; Robatzek and Somssich, 2001, 2002;

Gepstein et al., 2003; Lin and Wu, 2004; Buchanan-Wollaston et al., 2005). Leaf sags have been

reported in some plants such as maize (Smart et al., 1995), barley (Kleber-Janke and Krupinska,

1997), rice (Lee et al., 2001), Arabidopsis thaliana (Lohman et al., 1994), tomato (John et al.,

1997).

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Photosynthetic responses of annual plants can be improved by extending duration of heat

stress. This can be achieved by delaying the senescence leaf (Thomas and Howarth, 2000). Stay

green (SGR) proteins are responsible for the green-flesh and chlorophyll retainer during

senescence. Stay green (sgr) is upregulated that delays senescence (Buchanan-Wollaston, 1997;

Nam, 1997). In addition to Triticum durum, sgr expressed in other cereals such as sorghum,

maize, rice (Tao et al., 2000; Bekavac et al., 1995; Park et al., 2007; Buchanan-Wollaston,

1997). Stay-green regulates the loss of chlorophyll by disassembly of light-harvesting

chlorophyll-binding Protein (LHCP) during senescence (Park et al., 2007).

In view of the increasing adversaries due to global warming, it is necessary to find out

indicators and mechanisms of heat stress tolerance, which can be used to improve maize for

cultivation in warmer areas of the country. Ion homeostasis, relative membrane permeability and

production of H2O2, membrane per oxidation products and expression pattern of stress proteins

are important yardsticks to determine the heat stress tolerance of in maize. H2O2 accumulation is

also taken as the valid criterion for selection of high temperature stress tolerant materials.

Although many reports exists on the heat tolerance potential of maize, studies on the potential

indicators of heat tolerance under glasshouse conditions are lacking and need to be established

on firm grounds. These studies were conducted under the glass house conditions under the

following objectives:

1. Assessment of comparative heat stress response of maize at various phenological stages

during spring and autumn season

2. Determination of H2O2, accumulation of MDA, RMP and free amino acids in leaves as

indices of oxidative damage caused by heat stress

3. Establishment of possible relationships of the above attributes with heat stress tolerance

of maize genotypes

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CHAPTER-2 REVIEW OF LITERATURE

In nature, plants growing under unfavorable conditions such as salinity, drought, high

temperatures, freezing, low temperature, flooding, UV light, heavy metal, pathogenicity and

nutrient deficiency may display delayed growth and development, reduced productivity and, in

extreme cases, plant death. Plants display distinct changes in the morphological features in

response to these abiotic stresses, including shortened of life cycle (Porter, 2005). In some plants

these morphological changes occur to overcome or reduce the harmful effect of stresses

(Ferguson, 2004; Wang et al., 2004).

High temperature stress is a great modulator of growth and productivity (Zhang et al.,

2000). Prevailing high temperature reduces crop yield and affect plant growth from germination

up to maturity. The mechanisms leading to the survival of a crop under heat stress entail changes

in physiology and accumulation of osmolytes like proline, glycinebetaine, soluble sugars and

proteins (Wahid and Close, 2007; Verbruggen and Hermans, 2008). In addition, recent studies

show that changes in the expression levels of an array of genes are major mechanisms that

contribute to our understanding of heat stress responses of plants (Hazen et al., 2003). An

account of pertinent literature reflecting the effect of high temperature on various aspects of plant

growth and development is given below:

2.1 Plant growth and phenology

High temperature is a major determinant of agricultural production throughout the world and its

effects are evident at all critical growth stages starting from seed germination to seed yield. An

account of changes in the phenology of plants is elaborated underneath.

2.1.1 Seed germination and seedling survival

Seeds subjected to germinate in hot condition show reduced or even inhibited germination. A lot

of work has been done for improvement of seeds vigor, seedling, and reduce germination rate in

some field crops (Basra et al., 2005). High temperature effects on seed storage process and

kernel quality like starch, protein metabolism by effecting it’s enzymes at grain filling stage of

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maize (Wilhelm et al., 1999; Maestri et al., 2002). In studies on soybean, it was reported that

heat stress changed seed composition, seed protein expression profiles and reduce seed

germination and vigor, and thus appeared to determine the seed quality attributes (Egli et al.,

2005; Ren et al., 2009). Seed germination, seedling emergence and its establishment are

extremely affected by high temperature in a number of plant species (Grass and Burris, 1995;

Ashraf and Hafeez, 2004; Wahid et al., 2007). Columbo and Timmer (1992) demonstrated that

seedlings are more susceptible to high temperature stress than adult black spruce plant. Maize

shows climax germination and optimal growth at 20-30oC and 28-31oC, respectively (Hughes,

1979; Medany et al., 2007), and declines the coleoptiles growth in maize at 40oC and almost

ceases it at 45oC (Weaich et al., 1996; Akman, 2009). Many studies show that maize coleoptile

was more thermotolerant amongst all stages of seedling development (Venter et al., 1997;

Momcilovic and Ristic, 2007). Heat stress lowers the activity of specific enzymes and leads to

reduced synthesis of proteins in germinating maize embryos (Riley, 1981). The growth and

development of cotton (Gossypium hirsutum L.) seedling are reduced under high temperature

stress (Mahan and Mauget, 2005).

2.1.2 Vegetative growth and development

High temperature is a major environmental factor that determines the crop growth and yield in

some regions (Al-Khatib and Paulsen, 1999). Plant growth is often reduced at high temperature

(Blum, 1988). Plants grown under high temperature stress have lower biomass than those grown

at low temperature. Biomass, leaf area, photosynthesis and enzyme activities are decreased if

growth temperature is high (Kim et al., 2007). High temperature reduced stem elongation and

overall growth and limits plant survival (Ashraf and Hafeez, 2004). High temperature reduces

the plant growth by changing different plant mechanisms (Sibley et al., 1999). A steady increase

in temperature in each year may change cropping pattern, growth and economic yield of many

important crops (Wollenweber et al., 2003).

High temperature decreased the shoot dry weight, relative growth rate (RGR) and net

assimilation rate (NAR) in maize and millet (Ashraf and Hafeez, 2004) and sugarcane (Wahid,

2007). Heat shock affects the cell division in meristems and reduces the growth of various parts,

mainly the leaves (Salah and Tardieu, 1996). Maize leaf growth increased from 0 to 35oC, but

declined at 35 to 40oC. Above 40oC temperature, there was a severe reduction in photosynthesis

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and alteration in protein metabolism such as protein denaturation, aggregation, enzyme

inactivation, inhibited protein synthesis and its degradation (Dubey, 2005). Heat stress halted cell

wall elongation, stimulated cell division and altered cell differentiation (Potters et al., 2007).

High temperature influenced the leaf expansion, internode elongation, motivate the flower bud

abortion in Brassica napus (Young et al., 2004), which may be due to limited supply of water

and nutrients (Hall, 1992). Increased temperature affects growth, metabolism, development

(Rawson, 1995). High temperature causes photosynthetic acclimation and alters physiological

processes directly, and changes the pattern of development indirectly (Downton and Slatyer,

1972). It increased the rate of development and shortened the growth period in annual species by

virtue of rapid CO2 fixation and biomass production before setting seed (Rawson, 1992;

Morison, 1996). High temperature decreased the growth and accumulation of starch in tubers

greater than shoot but did not affect the glucose in potato tubers (Lafta and Lorenzen, 1995).

2.1.3 Reproductive growth and yield attributes

High temperature greatly affects the reproductive growth by increasing flower abortion and

decreasing seed size (Talwar et al., 1999). Shah and Paulsen (2003) demonstrated that

photosynthesis and leaf area, shoot, grain biomass and sugar contents of kernels are rapidly

decrease under high temperature. Pollination, an important stage in reproductive development, is

especially sensitive to heat stress; the mature pollens being more sensitive failed to fertilize

(Dupuis and Dumas, 1990). High temperature stress causes premature development of anther and

arrests its cell proliferation (Oshino et al., 2007). It interferes with pollen and anther

development, and causes male sterility in certain plant species (Sakata et al., 2000; Sato et al.,

2006; Abiko et al., 2005). Heat stress affects kernel development in maize plant with the

accumulation of ZEIN transcript during cell division (Monjardino et al., 2006). It reduced the

rate of dry matter accumulation, kernel density and reproductive growth in maize, wheat and

Suneca during kernel development and its filling (Wilhelm et al., 1999; Maestri et al., 2002).

Kernel dry weight reduced from 79 to 95% in field conditions in B-73 inbred line of maize under

heat stress (Commuri and Jones, 2001). High temperature effects the endosperm development in

maize and reduces grain yield during endosperm cell division. These effects were due to dry

matter accumulation, interruption of cell division, aberrant sugar metabolism and starch

biosynthesis in endosperm of kernels (Monjardino et al., 2005).

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2.2 High temperature and physiological phenomena

Long-term or even short-term exposure to high temperature culminates in changed metabolic

functions (Wahid et al., 2007). These alterations include water and nutrient uptake,

photosynthesis membrane properties, osmotic relations and gene expression. Accounts of these

phenomena are described underneath.

2.2.1 Water relations

Water is a basic need from seed germination to plant maturation. Heat stress leads to increased

evapo-transpiration of water from the plant surface and cause dehydration of aerial parts. High

temperature hampers the cell water relations and limits growth in many plant species (Machado

and Paulsen, 2001; Mazorra et al., 2002; Wahid and Close, 2007). Elevated temperature is

known to produce osmotic stress on the growing tissues due to reductions in root hydraulic

conductance and tissue water content (Jiang and Huang, 2001; Morales et al., 2003). Reddy et al.

(1991) demonstrated that reduction of water accessibility damage the growth and development of

cotton plants in sowing season. High temperature cause significant reduction in leaf growth of

sorghum (Sorghum bicolor) and water status and water potential in wheat leaves (Shah, 1992).

Low leaf water potential affects the nitrogen and protein metabolism to a great extent (Lawlor

and Cornic, 2002; Molnar et al., 2002).

High temperature hampers water, ions and organic solutes movement across the

biological membranes. It affects with photosynthetic and respiratory processes (Taiz and Zeiger,

2006), increases evapo-transpiration rate (Tsukaguchi et al., 2003) and reduces the leaf osmotic

potential and increases the leaf fluorescence (Huve et al., 2005). Heat stress induces the closure

of stomata and negatively influences leaf water status (Berry and Bjorkman, 1980). High

temperature induced water stress is closely associated to reduction of soil water contents (Talwar

et al., 1999).

2.2.2 Ionic and nutrient relationships

Mineral nutrition acquisition and assimilation is strongly influenced by high temperature stress in

plants (Taiz and Zeiger, 2006). Some essential nutrients such as carbon (C), nitrogen (N),

calcium (Ca), magnesium (Mg), phosphorus (P) and sulfur (S) are structurally important for the

proteins, nucleic acids, chlorophylls, certain secondary metabolites and defense related micro-

10

and macromolecules, while others have both structural and functional roles (Epstein and Bloom,

2005; Taiz and Zeiger, 2006). High temperature raised the rate of physiological processes of

plant growth, which determine nutrient absorption (Tollenaar, 1989). Under high temperature,

plants explore larger volumes of soils to absorb more P from the root (Fohse et al., 1988). K and

P uptake increased significantly with increase in temperature and maximum uptake of both was

evident at 32 and 38°C in maize roots, respectively (Bravo and Uribe, 1981). Under high

temperature stress, N concentration decreased sharply and S concentration decreased slightly,

while sodium (Na) was not affected (Muldoon et al., 1984).

Studies showed that the pattern of diurnal uptake of nitrate and dry matter accumulation

in maize seedling at varying day/night temperature of 30/20, 30/30, and 35/35°C. A greater

nitrate uptake took place at 30/30oC (Polisetty and Hageman, 1989). High temperature caused a

significant decrease in the shoot dry mass, RGR and NAR (Wahid, 2007). Significant increase in

the uptake of Ca and P and decrease one of N, S, Mg and Na was found at high temperature

(Ashraf and Hafeez, 2004). In sorghum, comparison of various temperatures for the uptake of

certain nutrients indicated that N and P was the highest at 27oC in whole plant, leaf, stem and

root (Ercoli, 1996).

High temperature affects the rate of biochemical reactions and enzyme denaturation,

resulting in decreased enzyme activities (Fukuokan and Enomoto, 2002). Nitrate reductase (NR)

is an important enzyme, the activity and stability of which increases by hardening (hyperthermia)

against a number of inactivating factors such as heating, proteolysis, in vitro and in vivo enzyme

degradation and enhances its ability to repair heat stress induced injury (Taiz and Zeiger, 2006).

The thermal stability of NR increased only in seedlings that were hardened at 40 and 44oC. A

short term exposure to heat stress led to recover the functional activity of NR without de novo

synthesis of the enzyme protein (Lyutova and Kamentseva, 2001). After absorbing nitrate, NR

controls the rate of protein synthesis (Srivastava and Naik, 1980). The activity of NR is affected

by light in several ways. Light activates one or both chloroplast photosystems in green tissues

and increases transport of stored NO3- from vacuole to cytosol where reduction of NR occurs

(Granstedt and Huffaker, 1982). Secondly, light activates the phytochromes, which increases the

potential of the ribosome to synthesize various proteins. Thirdly, light inactivates various

proteins, which act as inhibitors of NR activity. Finally light increases NR activity by increasing

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the carbohydrate supply; NADH required for nitrate reduction is produced from these

carbohydrates when they are respired (Aslam and Huffaker, 1984).

2.2.3 Osmolytes accumulation

Certain organic osmotica such as leaf soluble proteins, proline and soluble sugar are important

adaptive components of heat tolerance in many plant species to prevent water loss and in

mediating osmotic adjustment (Ashraf et al., 1994; Sakamoto and Murata, 2002; Wahid and

Close, 2007). These osmolytes include nitrogenous compounds such as quaternary ammonium

compounds (QACs), and tertiary sulphonium compounds, other amino acids like proline, aspartic

acid and glutamic acid (Samuel et al., 2000; Wahid et al., 2007), polyamines, glycine betaine,

ectoine, polyols and soluble sugar (Ashraf et al., 1994; Wang et al., 2003; Chen and Murata,

2002; Rontein et al., 2002; Sairam and Tyagi, 2004; Wahid and Close, 2007). Accumulation of

proline occurs in many organisms subjected to abiotic stresses including heat stress (Saradhi et

al., 1995; Siripornadulsil et al., 2002; Verbruggen and Hermans, 2008).

Proline and QACs, in addition to being N-rich, accumulate in plants under variety of

environmental stresses, which may decrease stress-induced cellular acidification or primary

oxidative respiration to give required energy for survival. Accumulation of proline under stress

shows association with stress adaptation in higher plants (Lalk and Dorffling, 1985; Bartels and

Sunkar, 2005; Knipp and Honermeier, 2006). The proline accumulation is more important than

osmotic adjustment and stored carbon and nitrogen (Hare and Cress, 1997; Hare et al., 1999).

High level production of proline during stress may maintain NAD(P) + NAD(P)H ratios, which

matched well with metabolic steps under normal condition (Hare and Cress, 1997; Foreman et

al., 2003). At 35oC, there were high levels of proline and choline in tomato plants (Rivero et al.,

2004). Proline accumulation is controlled by tissue water status and unchanged by tissue

temperature up to 39°C in barley (Chu et al., 1974). Irigoyen et al. (1992) found a rapid

accumulation of proline with declined tissue water potential in alfalfa. High level of proline also

reduces the generation of free radical due to osmotic stress (Hong et al., 2000). Thus proline and

GB-synthesis may defend cellular redox potential under high temperature (Alia et al., 1998). A

rapid elevation of free proline was found in the sense-transforments that exhibited the smallest

amount of H2O loss, while the slowest elevation of proline levels was detected in antisense-

transforments that exhibited the greatest H2O loss during stress.

12

Genetic manipulations of osmolytes like proline levels also affect the stress induced

changes in the concentration of several other amino acids, which show the coordinated regulation

of metabolic pathways (Simon-Sarkadi et al., 2005). Combined effect of heat and drought stress

exhibited an increase in proline concentration in cotton (De Ronde et al., 2000). Accumulation of

free proline under stress conditions does not inhibit biochemical reaction and play an

osmoprotectant function during osmotic stress (Sawahel and Hassan, 2002). In root of maize

plant, solute potential declined primarily due to proline and soluble sugars accumulation

(Rodríguez et al., 1997). In A. thaliana, two pyrroline -5- carboxylate synthetase (P5CS) genes,

which are the rate limiting enzymes, play important roles in proline biosynthesis (Szekely et al.,

2008). These enzymes are regulated by transcriptional regulation and feedback inhibition in

plants (Zhang et al., 1995). Feedback regulation of P5CS plays a function in regulating the level

of proline in plants. Accumulation of proline is the component of stress signal that manipulate

the adaptive responses (Maggio et al., 2002), and in plant parts such as pollen grains, seeds and

roots, but is dependent upon the plant age, leaf part, leaf position on the plant and leaf age

(Chiang and Dandekar, 1995). Free proline accumulation may scavenge reactive oxygen species

(ROS) by enhancing photochemical electron transport activities (Smirnoff and Cumbes, 1989;

Pinheiro et al., 2001), maintaining enzyme structure and activity (Rajendrakumar et al., 1994;

Samuel et al., 2000) and protection of membrane integrity as an adaptation to water deficiency

(Hare, 1995; Bohnert and Jensen, 1996; Ashraf and Foolad, 2007).

Soluble sugar also plays important roles in osmotic regulation of cells under heat stress

(Bolarin et al., 1995). Expression of some seed germination genes is controlled by the sugars

levels (Reynolds and Smith 1995; Yu et al., 1996). Accumulation of sugars in mature seeds is

essential for the improvement of desiccation tolerance (Hoekstra et al., 2001). Study on 11

enzymes of carbohydrates metabolism from developing endosperm showed that ADP glucose

pyrophosphorylase, glucokinase, sucrose synthase and soluble starch synthase were highly

susceptible to the high temperature stress. Prolonged heat stress affects seed storage processes in

many maize inbreds (Wilhelm et al., 1999).

2.2.4 Leaf gas exchange

All aspects of photosynthesis are prone to episodes of high temperature. Increased ambient

temperature affects plant productivity by damaging photosynthesis (Al-Khatib and Paulsen,

13

1990). The maize seedling grown at 25oC and transferred to 35oC for 20 min led to 50%

inhibition in photosynthesis (Sinsawat et al., 2004). In maize, high temperature caused a distinct

decrease in growth, transpiration, respiration and photosynthesis (Karim et al., 2000), and final

yields (Stone, 2001). The net photosynthesis in maize was maximum near 31oC, which decreased

at temperature above 37oC while completely inhibited near 45oC (Crafts-Brandner and Salvucci,

2002). High temperature inhibited net photosynthetic (Pn) and stomatal conductance

significantly in many plant species (Ranney and Peet, 1994; Crafts-Brandner and Salvucci, 2002;

Morales et al., 2003; Ashraf and Hafeez, 2004). Net photosynthesis (Pn) of developed and nearly

developed leaves was more sensitive than developing leaves (Karim et al., 1997, 1999).

Photosynthetic apparatii are highly sensitive to high temperature and are inhibited when

leaf temperature exceed 38oC. This is because C4 plants have better ability to photosynthesize

under higher temperature than C3 plants (Berry and Bjorkman, 1980; Edwards and Walker, 1983;

Wahid and Rasul, 2005). It declines the activation of rubisco, a highly susceptible components of

the photosynthetic apparatus in C3 as well as C4 plants (Crafts-Brandner and Salvucci, 2000,

2002; Law and Crafts-Brandner, 1999; Salvucci and Crafts-Brandner, 2004). Photosystem (PS)

II, water splitting and oxygen evolving complex (OEC) in photosynthesis are more heat sensitive

components of photosynthesis (Edwards and Baker, 1993; Pastenes and Horton 1996a;

Heckathorn et al., 1998a). Leaf high temperature can disrupt the ultra structural characteristic of

chloroplast (Ristic et al., 2004). Thylakoid lamellae and stroma of chloroplast are very much

sensitive to high temperature (Wise et al., 2004). High temperature inhibits photosynthesis by

affecting the structure of thylakoid lamellae (Karim et al., 1997). Extensive studies show that

both PSI and PSII are damaged by increased high temperature. In barley and potato, heat stress

damaged PS-I and PS-II, and affected electron transport, which is very important during

photosynthesis (Havaux, 1998; Szilvia et al., 2005). Heat stress damaged the antenna complex of

PSII and reduced the respiratory and photosynthetic behavior (Carpentier, 1999; Zhang et al.,

2005). High temperature during greening led to the inactivation of PSI and PSII (Sasmita and

Narendranath, 2002). Heat stress inhibited the activity of PSII as determined from electron

transport measurement (Rokka et al., 2000).

The photosynthesis in C3 plants is more affected by high temperature than C4 plants

(Wahid and Rasul, 2005). Increased temperature reduced the activation of rubisco in the exposed

leaf tissue and increased the level of rubulose-1, 5-bisphosphate (Feller et al., 1998; Crafts-

14

Brandner and Law, 2000). High temperature altered the energy sharing by changing the action of

Calvin cycle and other metabolic processes such as photorespiration, and synthesis and stability

of the rubisco enzyme (Pastenes and Horton, 1996b), disruption of electron transport activity and

bound RUBP supply by heat stress (Ferrar et al., 1989). High temperature inhibited the activity

of rubisco by transfer of rubisco activase gene, which result inhibition of photosynthesis as

compared to control plants (Sharkey et al., 2001). High temperature enhanced chlorophyllase

activity and decreased photosynthetic pigments concentrations (Todorov et al., 2003). The loss

of chlorophyll is a good indicator of heat tolerance in wheat (Ristic et al., 2007; 2008). High

temperature enhanced chlorophyll a:b ratio and declined chlorophylls-to-carotenoids ratio in

sugarcane (Wahid, 2007).

2.2.5 Cell membrane thermostability

The cellular membranes play an important function in maintaining integrity of cell, by involving

in signal transduction and ion homeostasis under environmental stresses (Kaur and Gupta. 2005;

Tuteja and Sopory. 2008). High membrane stability, determined in terms of changes in ion-

leakage, is taken as an index of heat tolerance in several grain, forage and Pasteur crops

(Saadalla et al., 1990; Blum et al. 2001; Ashraf et al., 1994; Marcum, 1998; Ismail and Hall,

1999; Wahid and Shabbir, 2005). The membrane stability and its functions are susceptible to

high temperature (Nishida and Murata, 1996; Wahid et al., 2008). Maize showed a great

reduction in membrane stability under high temperature (Yang et al., 1996). Plant processes such

as photosynthesis, respiration, assimilate partitioning etc. are directly impinged by high ambient

temperature (Tsukaguchi et al., 2003; Iwaya-Inoue et al., 2004). Quite discernible changes occur

in the cellular membranes of organelle (Nash et al., 1982; Dionisio-Sese et al., 1999; Wahid et

al., 2007)

High temperature induced injury to thylakoids in winter wheat (Ristic et al., 2007),

resulting in the production of reactive oxygen species (Camejo et al., 2006; Guo et al., 2006).

Finding genotypic differences for heat tolerance based on leaf electrolyte leakage may be more

effective with plant subjected to heat stress (Li et al., 1991). High temperature damages

membrane by lipid peroxidation (Bhattacharjee and Mukherjee, 1998) making them more

permeable to ions (Wen-yue et al., 2001). Measurement of electrolyte leakage indicates the stress

damage to assess the harshness of existing stress (Foyer et al., 1997). Cell membrane

15

thermostability (CMT) is used to identify the genetic differences for high temperature tolerance

in sorghum and wheat germplasm (Sullivan and Ross, 1979; Ibrahim and Quick, 2001). Agarie et

al. (1998) used electrolyte leakage as an effective tool for measuring CMT in root tissues, which

exhibited a great sigmoid response to temperature for this tissue. Saadalla et al. (1990) found an

association in membrane thermostability (MT) between seedlings and flag leaves at anthesis for

genotypes of wheat grown under control and high temperature. Sullivan (1972) prepared a heat

tolerance analysis to demonstrate CMT by measuring the quantity of electrolyte leaked from leaf

after exposure to high temperature stress.

High temperature effect on cell MT is associated with yield performance (Rahman et al.,

2004). Agarie et al. (1995) used the cell membrane stability (CMS) as heat tolerance test in rice.

Injury to membranes from sudden heat shock may result from either denaturation of proteins or

increases unsaturation of fatty acids, leading to membrane rupture and loss of cellular solutes

(Savchenko et al., 2002). Genetic difference in cellular thermotolerance expressed by CMT of

membrane is a site of primary physiological injury with high temperature (Fokar et al., 1998;

Blum, 1988). MT is heritable and shows significant genetic association with yield (Fokar et al.,

1998). Relative cell injury (RCI) is a determinant of cellular and/or tissue heat tolerance. A lower

RCI reflects high CMT and vice versa (Rahman et al., 2004). Damage due to stress to plasma

membranes was much lesser in younger than the older maize leaves (Karim et al., 1999).

Excessive dehydration from the leaf surface due to heat stress leads to the disruption of cell

membranes by solublization and peroxidation of membrane lipids (Wen-yue et al., 2001; Jiang

and Haung, 2001; Iba, 2002).

2.2.6 Oxidative damage

Like other abiotic stresses, high temperature also induces oxidative stress as a result of an

imbalance in the formation and metabolism of ROS (Lee et al., 1983; Dat et al., 1998; Sairam

and Tyagi, 2004). Chloroplast, mitochondria, endoplasmic reticulum and microbodies are major

sites of ROS and malondialdehyde (MDA) production under abiotic stress (Foyer et al., 1997;

Dat et al., 1998; Breusegem et al., 2001; Sairam and Srivastava, 2002; Apel and Hirt, 2004;

Kukreja et al., 2005). Various roles have been assigned to ROS in plant development. There is

proof that ROS are necessary for growth of root hair, where they manage the activity of Ca2+

channels necessary for polar growth (Rachel and Dolan, 2006). H2O2 is a regulator of gene

16

expression in the cells such as gene encoding antioxidant, cell defense, signaling, stress protein

and transcription factors (Hernandez et al., 2000; Gabriela and Foyer, 2002; Wahid et al., 2007,

2008). The H2O2 breaks the seed dormancy in conifers; enhance heat and salt tolerance in rice

(Uchida et al., 2002). However, with the production of various ROS e.g., superoxide radical (O-

2), hydroxyl radical (OH-), H2O2, and singlet oxygen (O21) in higher quantities, peroxidation of

membrane lipids and damage to important molecules such as nucleic acids, proteins, chlorophyll

and other important macromolecules takes place (Smirnoff, 1993; Scandalios, 1993; Foyer et al.,

1997; Sairam et al., 2000; Hernandez et al., 2000 and Foyer and Fletcher, 2001; Wahid et al.,

2007). A well known effect of high temperatures induced oxidative damage in plants is

imbalance in photosynthesis and respiration (Fitter and Hay, 1987). H2O2, an important ROS,

causes injury to cellular processes, reduces photosynthesis and enhances senescence (Dhindsa et

al., 1981). Under combined effect of heat and drought stresses, turf quality, relative water

contents, chlorophyll and protein content were reduced because of decreased activities of

antioxidant enzymes and increase in electrolyte leakage and membrane lipid peroxidation

(Huang and Gao, 1999; Jiang and Huang, 2000; 2001; Xu and Huang, 2004).

AOS levels increase in stressed tissue due to reduced antioxidant activity (Fadzillah et

al., 1996). Plants have well developed several enzymatic and non enzymatic antioxidant defense

systems as a line of defense to remove and detoxify intracellular structures (Noctor and Foyer,

1998; Liu and Huang, 2000; Fu and Huang, 2001; Alscher et al., 2002; Sairam and Tyagi, 2004;

Farooq et al., 2008). The enzymatic antioxidants include catalase (CATs), superoxide dismutase

(SOD), glutathione peroxidase such as ascorbate peroxidase (APX), glutathione reductase (GSH)

and glutathione-synthesizing enzymes (Arora et al., 2002; Sairam et al., 2002; Gong et al.,

2005). SOD scavenges the superoxide radical to H2O2, which is converted to water and oxygen

by the action of glutathione peroxidase or CAT and GHS reductase in chloroplast and

mitochondria respectively (Foyer and Halliwell, 1976; Scandalios, 1993; Anderson, 2002). CAT

is necessary for the elimination of H2O2 generated in the peroxisomes during photorespiration

(Noctor et al., 2000). These enzymes rapidly demolish huge amounts of H2O2, but at the same

time they allow low levels to continue probably to retain redox signaling pathways (Noctor and

Foyer, 1998).

17

2.3 Genes and proteins expression

Most of the crop plants respond to stress situations by inducing the expression of genes and

synthesis of stress proteins (Schlesinger et al., 1982; Lindquist and Craig, 1988). In higher

plants, expression of genes and synthesis of stress and related proteins have been extensively

studied (Vierling, 1991; Rahman et al., 2002; Wahid et al., 2007). An account of literature on

this aspect under heat stress is reviewed below.

2.3.1 Gene Expression and high temperature

Transcriptional regulation play an important role in plant defense from abiotic stresses including

heat stress (Singh et al., 2002). Transcriptional analyses show that high temperature induces

numerous genes encoding transcriptional factors, which are involved in heat stress response and

tolerance (Chen and Zhu, 2004; Kotak et al., 2007). Modification of gene expression improved

stress tolerance in Arabidopsis and rice. The synthesis and accumulation of HSPs through HSFs

network play an important role in plant heat tolerance (Wang et al., 2004; Nakamoto and Vigh,

2007; Kotak et al., 2007) Upon exposure to high temperature, pattern of gene expression is

changed, which is pivotal for thermotolerance (Yang et al., 2006). Amounts of specific mRNA

synthesis, mRNA stability, translation efficiency and alteration in protein activity increase in

plants as results of gene expression (Sullivan and Green, 1993). All organisms synthesize heat

shock proteins (HSPs) in small numbers, when suddenly exposed to high temperature. It is found

that expression of specific gene is required when reproductive organ of plant are exposed to

abiotic stresses (Sorensen et al., 2002; Yamaguchi-Shinozaki and Shinozaki, 2000; Shinozaki et

al., 2003; Chinnusamy et al., 2004). Heat stress altered gene expression in reproductive organ of

plant (Dupuis and Dumas, 1990; Oshino et al., 2007). Altered development and demarcation of

pollen mother cell due to heat shock is due to tissue specific alterations in gene expression

(Sakata et al., 2000; Abiko et al., 2005). Pollen development and pollen tube growth in maize are

more susceptible to heat stress (Gagliardi et al., 1995; Mascarenhas and Crone, 1996).

Heat shock protein 70 (HSP70), a widely expressed protein, and heat shock factor (hsf)

gene is elevated during heat shock at earlier pollen development in maize. Hsfs are essential for

gene expression in response to high temperature (Nover et al., 2001; Baniwal et al., 2004).

Various studies show that distinctive hsp genes are not expressed in germinating pollen. Only

hsp18 and hsp70 genes are transcribed in response to heat shock. Hopf et al. (1992) reported that

18

the defective heat shock response of mature pollen of maize was due to reduced induction of heat

shock gene transcription. HSP70 assisted in translocation, proteolysis, protein translation, protein

folding, aggregation and refolding of denatured proteins (Zhang et al., 2005). Some reports

showed that HSP70 assists in ATP unfolding, folding, intracellular distribution, assembly,

degradation of proteins and prevent the protein denaturation during high temperature stress (Iba,

2002; Weggle et al., 2004; Gorantla et al., 2007).

Different studies revealed that several genes are up and down regulated by abiotic and

biotic stresses (Kawasaki et al., 2001; Nogueira et al., 2005). Elevated temperature affects the

gene expression in storage protein synthesis and starch metabolism during grain filling stage in

rice (Yamakawa et al., 2007). This effect down regulates the several protein or starch synthesis

related enzyme proteins such as GBSSI, BEIIb and prolamin (13-kD), and up regulates the

enzymes such as α- amylase genes and HSPs (Lin et al., 2005; Yamakawa et al., 2007). Recently

it is reported that α-amylase genes in seeds of rice reduced the seed weight and chalkiness during

ripening under heat stress (Asatsuma et al., 2006; Yamakawa et al., 2007). In carrot, different

genes such as tubulin gene, hsp90 gene, oleosin gene, a LEA gene, DC8 and DC59 genes were

observed in embryonic heat-shock cell lines. High temperature declined the expression of a LEA,

an oleosin, DC8 and DC59 genes (Hatzopoulos et al., 1990; Milioni et al., 2001).

Recently, transcriptome studies identified many stress-responsive genes and encoding

transcriptional factors during environmental stresses. Changing the expression pattern of

transcriptional factor may also greatly manipulate the stress tolerance in plant (Chen et al.,

2002). Plants practice oxidative stresses during primary adaptation stage to a stress. Definite

responses require continued expression of many genes relating to processes definite to individual

stresses (Sung et al., 2003). Recent transgenic approaches may modify the heat tolerance in

plants which is a multigenic characteristic. This multigenic phenomenon, modifying the

expression pattern of transcription factors, motivates a series of genes (Dong et al., 2003). In

Arabidopsis, a new HSF3 that depressed in response to heat-shock and over expression of it

enhanced the thermotolerance and increased the activity of APX in transgenic plants (Schöffl et

al., 1999; Panchuk et al., 2002). Similarly Hot1-Hot4 gene may function as heat shock protein

and abolished acquired thermotolerance phenotypically; Hot1 is Hsp101 in A. thaliana (Hong

and Vierling, 2000). HsfA1 acting as crucial regulator of thermo-tolerance in tomato that

reduced the expression of heat shock genes in co-suppression lines (Mishra et al., 2002). Heat

19

shock induces many genes which are attributed to heat shock elements (HSE).These heat-shock

elements (HSE) are situated in the promoter region of hsp genes (Hubel and Schöffl, 1994).

Transgenic expression confirmed that Heat-shock transcription factor (HSF) binding to

pentameric nucleotides (5`-nGAAn-3`) of HSE sequences (Perisic et al., 1989; Sung et al., 2003)

and this HSF-HSE interaction and transcriptional activation is very conserved in nature. In HSP

gene expression of maize, Ca2+ and calmodulins (CaM) are playing important role in the

presence of the HSF. These HSF are induced by heat-shock. The activity of these factors is

inhibited at high temperature (Li et al., 2004).

The primary characteristic of plant thermometer has not been recognized. It has been

recommended that alteration in membrane fluidity can manipulate the gene expression.

Membrane fluidity acts a key role in temperature sensing (Over et al., 2000). In Synechocystis,

hsp17 gene act as a fluidity gene and membrane controls signaling mechanism in response to

high temperature stress (Horvath et al., 1998). A model for temperature sensing and regulation of

heat stress response integrates the observed membrane alterations. It is recommended that

thylakoid membrane acting as temperature sensor, is physiological pertinent (Horvath et al.,

1998; Sung et al., 2003). Many genes are concerned with signaling pathways and encode

proteins particularly mitogen activated protein kinase (MAPK), histidine kinase, Ca2+ dependent

protein kinase (CDPK), SOS3, Ca2+ sensor family and also various transcription factors are

regulated under various biotic and abiotic stresses (Ichimura et al., 2000). Cytosolic Ca2+ sharply

rises in response to high temperature stress (Larkindale and Knight, 2002), which is related to the

acquired thermotolerance. Kaur and Gupta, (2005) demonstrated that high temperature

transduced the signals to MAPK by cytosolic Ca+2 influxes, and eventually up regulated the

calcium dependent (CDP) kinase. MAPKs may function as signaling conveys to transmit

information within cell. It is reported that the induction of uptake of Ca2+ and some calmodulin

related genes induced thermotolerance in maize seedling (Gong et al., 1997a, b).

2.3.2 Heat shock proteins

Synthesis of HSPs is a general response to temperature stress (Vierling, 1991; Parsell and

Lindquist, 1993). However, the physiological functions of HSPs are not yet clear (Lee et al.,

1994). The HSPs are induced by high temperature at any stage of development in plants. The

accumulation of small heat-shock proteins (sHSPs) in response to elevated temperatures has been

20

reported in wheat (Weng and Nguyen, 1992), pea (Lenne and Douce, 1994), corn (Nieto-Sotelo

et al., 1990), Chenopodium album and tomato (Downs et al., 1999), and common bean (Simões-

Araújo et al., 2003). They play a crucial role in protecting the photosynthetic system from heat

damage. Carrot cells exposed to 38°C resulted in gradual termination of normal protein synthesis

twined with an induction of new class of HSPs (Pitto et al. (1983). HSPs synthesis has been

correlated with differences in thermotolerances of different lines of sorghum (Ougham and

Stoddart, 1986), and unique HSPs were associated with thermotolerant in wheat (Nguyen et al.,

1989). Clarke and Critchley (1990) compared two cereals showing greater diversity in HSPs that

were induced in the subtropical C4 species (sorghum) compared to temperate C3 species (barley).

Heat tolerance in maize was triggered by shorter heat shock or gradually increased temperature

(Gong et al., 2001), which was associated with the synthesis of a new activase polypeptide

(Crafts-Brandner and Salvucci, 2002).

High temperature induces generally low molecular weight heat shock proteins (Wahid et

al., 2007). In plants, a heat shock of 8 to 10°C above ambient temperatures induces the synthesis

of both high (60 to 110 kDa) and low (15 to 30 kDa) molecular weight HSPs (Vierling, 1991;

Waters et al., 1996; Sun et al., 2002). These HSPs were induced either to protect the plant from

injury or to help repair the injury caused by the heat stress (Leshem and Kuiper, 1996). The

synthesis of HSPs occurred in different plant species when they were exposed 10-15oC above

growing temperatures (Dubey, 1999). Their synthesis is extremely fast, diverse and intensive in a

variety of organisms (Parsell and Lindquist, 1993; Wahid et al., 2007).

Both cytosolic and organelle synthesis of HSPs has been well studies and reported. Some

HSPs were accumulated in the cytosol and chloroplast at 27, 43 and 37oC respectively, which

appeared to play a role in photosynthesis and thermotolerance (Heckathorn et al., 1998b). In

maize, high temperature caused the accumulation of chloroplast protein synthesis elongation

factor EF-TU, which defended the chloroplasts proteins from heat-induced damage (Ristic et al.,

2004; Momcillov and Ristic, 2004). Maize EF-TU is a 45-46 kDa HSP, confined to chloroplast

stroma, is involved in development at heat tolerance in maize (Ristic et al., 1998; Bhadula et al.,

2001; Moriarty et al., 2002). In maize, a heat shock of 40°C induces the synthesis of HSP with

molecular weight of 98 kDa (Nieto-Sotelo et al., 1990). The shsps genes are found in distinct

plant compartments like cytosol (Class I and Class II), chloroplast, ER, mitochondria and

membranes (Waters et al., 1996), which are encoded by six nuclear gene families. Interaction of

21

HSPs 22kDa with the C. album and common bean chloroplast membranes affects the

composition of the membrane and decreases its fluidity and increases the efficiency of ATP

transport (Barua et al., 2003; Simões-Araújo et al., 2003).

Mitochondrial HSP have been isolated from pumpkin (Cucurbita pepo) cotyledons,

which are produced under heat stress (Tsugeki et al., 1992; Kuzmin et al., 2004). They act as

molecular chaperones in vitro (Schöffl et al., 1999; Guo et al., 2001; Kim and Schöffl, 2002;

Port et al., 2004), prevent aggregation of denatured proteins (Sheffield et al., 1990), help in

folding of newly synthesized polypeptides, refolding of denatured proteins (Lee et al., 1994;

Goloubinoff et al., 1999), or re-solubilize aggregated proteins (Parsell et al., 1994). HSP68

synthesis was restricted to mitochondria as a precursor protein, but its synthesis increased during

heat shock in cell of plant species studied (Neumann et al., 1993). When wheat, maize and rye

seedling were exposed at 42oC temperature, five mitochondrial LMW HSPs (19, 20, 22, 23 and

28 kDa) were induced in maize and only one (20kDa) in rye and wheat mitochondria; the

tolerance of maize was higher than wheat and rye (Korotaeva et al, 2001). The specific HSPs

have been identified in potato, maize, soybean, barley and tomato (Neumann et al., 1993), peas

(Ko et al., 1992), which are encoded by nucleus (Watts et al., 1992) under high temperature.

Some putative functions have been assigned to HSPs produced under normal or high

temperature environment. The rapid accumulation of HSPs could play a significant role in the

safety of the metabolic apparatus of the cell. Some HSPs are produced in some developing cells

under control condition (Hopf et al., 1992) and their expression in plant is limited to

embryogenesis, germination, pollen development and pollination, fruit set and its maturation

(Vierling, 1991; Sun et al., 2002; Prasinos et al., 2005). HSPs were produced in greater amounts

in etiolated maize seedling after 5 h under high temperature stress (Lund et al., 1998). Acquired

thermotolerance depends upon the synthesis of HSPs and cellular localization (Heckathorn et al.,

1999; Korotaeva et al., 2001). In arid and semi arid regions, dry land crops may produce and

accumulate significant amount of HSPs in response to elevated leaf temperatures. In 2 day old

soybean seedlings, HSPs appeared to contribute to maintain the conformation of other protein

structures, which may be necessary for the acquired thermo-tolerance (Jinn et al., 1997). The

wide diversification and abundance of HSPs in plants important alteration to temperature stress

(Waters et al., 1996). A distinct group of HSPs was induced in male tissues of maize under heat

22

stress. In contrast, the mature pollen was susceptible to high temperature and pollen viability was

extremely reduced due to no or reduced synthesis of HSPs (Dupuis and Dumas, 1990).

HSP101 in various plant species has dual properties i.e., thermotolerance as well as

translational activator (Chang et al., 2007; Wells et al., 1998). It plays a central role in heat

tolerance to extreme conditions in Arabidopsis (Hong and Vierling, 2000; Queitsch et al., 2000).

High levels of HSP101 were present in developing and mature grains of wheat, maize and rice

(Singla et al., 1998). In one study, HSP101 had no harmful effects on development of plant if

persisted in the absence of stress (Queitsch et al., 2000; Hong and Vierling, 2001; Nieto-sotelo et

al., 2002). In another study, HSP101 reduced the growth rate of primary root of maize (Nieto-

sotelo et al., 2002). HSP104 belongs to protein family of Hsp100/Clp (Schirmer et al., 1996),

plays a great role in acquired heat tolerance in yeast (DeVirgilio et al., 1994) and is used in the

renaturation of cumulative protein, Hsp70 and Hsp40 (Glover and Lindquist, 1998).

2.3.3 Other heat induced proteins

Besides HSPs, many other heat induced proteins including ubiquitin manganese peroxidase,

cytosolic Cu/Zn-superoxide dismutase and dehydrins (DHNs) are expressed (Sun and Callis,

1997; Wahid et al., 2007). These proteins play a crucial role in protein degradation pathway and

oxidation stress response (Schöffl et al., 1999).

a. Dehydrins

LEA proteins are classified into three groups (Dure et al., 1989), and DHNs belong to subclass

of LEA group II. DHNs are produced at the later stages of seed development in various plant

species under drought, salinity, low temperature, heat stress, nutrients deficiency and ABA

application (Close, 1996; Campbell and Close, 1997; Svensson et al., 2002; Pulla et al., 2007;

Wahid and Close, 2007). D-11 from cotton (Baker et al., 1988), RAB16 (responsive to ABA) in

rice (Mundy and Chua, 1988) and RAB17 in maize (Vilardell et al., 1990) was cloned and

characterized by the first two DHN genes and later from both angiosperms and gymnosperms

(Campbell and Close, 1997; Ismail et al., 1999; Koag et al., 2003). Immunological evidence

indicated that DHNs are expressed in bacteria (cyanobacteria) (Close and Lammers, 1993), algae

(phaeophyta) (Li et al., 1997), liverworts (Hellwege et al., 1994), ferns (Reynolds and Bewley,

1993), ginkgo (Close and Lammers, 1993) and conifers (Jarvis et al., 1996). Using

23

immunological studies, DHNs were localized in the nucleus, cytoplasm, mitochondria,

chloroplasts and vacuole (Close, 1996; Campbell and Close, 1997; Wahid et al., 2007) and

associate to membranes system under abiotic stress (Koag et al., 2003). Immuno-

histolocalization studies indicated the DHNs distribution in the vegetative and reproductive

organs of several plant species under normal or stressful conditions. In maize, all parts of mature

embryos show dehydrins accumulation (Godoy et al., 1994). In recent studies, three low

molecular weight dehydrins were reported to be expressed in sugarcane leaves in response to

heat stress while all other conditions were same (Wahid and Close, 2007).

Structurally, dehydrins have three segments such as K-, S- and Y-segment (Close, 1996;

1997). The K-segment (EKKGIMDKIKEKLPG) is generally present in all dehydrins (Close,

1996, 1997; Svensson et al., 2002). The Y-segment (DEYGNP) presents in one to three copies

close to the N-terminal. All these segments are combined together in a regular way. The S-

segment along with K-segment regulates the activity of protein during phosphorylation (Plana et

al., 1991; Godoy et al., 1994). In dehydrins, Φ-segments form hydrogen bond with K-segments

resulting in the formation of amphipathic α-helix, which shows hydrophobic properties (Segrest

et al., 1990; Dure et al., 1989; Close, 1996). These Φ-segments are composed of proline, alanine-

rich and many polar and non polar amino acids. Dehydrins show considerable variations on the

basis of Φ-segments (Campbell and Close, 1997). Close (1996) identified five different types of

dehydrins such as Kn, Skn, KnS, Y2Kn and YnSk2 on the basis of structure by using the

notation “YSK Shorthand”.

Biochemical studies revealed that post-translational alteration may take place in DHNs.

For example, RAB17 from maize (Vilardell et al., 1990), DHN from tomato TAS14 (Godoy et

al., 1994) and DSP16 from Craterostigma plantagineum (Lisse et al., 1996) showed

phosphorylation. The phosphorylated peptide contains S-segment which regulates the activity of

those proteins (Plana et al., 1991).

b. Senescence associated genes

Temperature, pathogenic infection, drought, and nutrient deficiency; wounding and shading may

increase leaf senescence (Lohman et al., 1994; Oh et al., 1996; He et al., 2001; Liu et al., 2008).

About 183 senescence associated genes (sags) are involved in metabolism, energy metabolism,

gene expression regulations, protein biosynthesis regulations, pathogenicity, stress and flower

24

development (Liu et al., 2008). A number of encoding SAGs for proteinases such as serine

proteinase in parsley (Jiang et al., 1999), cysteine proteinase in Arabidopsis (Lohman et al.,

1994) and aspartic proteinase in Brassica (Buchanan-Wollaston and Ainsworth, 1997) are

associated with leaf senescence. Control of gene expression during senescence is complex. A

large number of SAGs and defense genes has been reported to express during leaf senescence in

some plants such as maize (Smart et al., 1995), barley (Kleber-Janke and Krupinska, 1997), rice

(Lee et al., 2001), A. thaliana (Lohman et al., 1994; Oh et al., 1996; Gepstein et al., 2003),

tomato (John et al., 1997; Drake et al., 1996), radish (Azumi and Wantanabe, 1991) and

Brassica napus (Buchanan-Wollaston and Ainsworth, 1997). High temperature accelerates the

senescence and results in decreased assimilation partitioning to grains (Spano et al., 2003).

Different stresses including high temperature induced the dehydration responsive genes

(ERD1), which is known as SAG15. This gene protects the cells from injury (Weaver et al.,

1998, 1999). A combine effect of heat-shock and drought induced a senescence associated gene

(SAG12) at least in Nicotiana tabacum which improved the stress tolerance in plants (Rizhsky et

al., 2002). Heat shock (40oC) induced tmr genes in Agrobacterium. These were transferred to

tobacco plants. The regulation of this gene was achieved by an inducible promoter, HS6871, to

control the activity of tmr gene from soybean, which delays the senescence (Smart et al., 1991).

Chen et al. (2002) identified 18 transcription factors such as WRKY genes in response to

senescence and environmental stresses including heat stress during plant growth and

development. These WRKY proteins improved the agronomic characters of crop plants. High

temperature disrupts the normal senescence. The initiation of premature senescence and

inhibition of certain thylakoid protein including LHCPII degradation occurred at elevated

temperature (Ferguson et al., 1993).

c. Stay green gene

Photosynthetic responses of annual plants improve by extending duration of vegetative growth.

This can be achieved by delaying leaf senescence (Thomas and Howarth, 2000). Stay-green

(Sgr) proteins are responsible for the green-flesh and retention of chlorophyll during senescence

(Park et al., 2007; Barry et al., 2008). The trait stay-green is divided into five types such as type

A, B, C, D and E based on chlorophyll retention during leaf senescence (Thomas and Smart,

1993; Thomas and Howarth, 2000). A stay-green protein typically down regulates the

25

chlorophyll degradation at the transcriptional level resulting in delayed senescence (Buchanan-

Wollaston, 1997; Nam, 1997; Park et al., 2007). Delaying leaf senescence by only two days,

result about 11% increased carbon fixation in Lolium temulentum (Thomas and Howarth, 2000).

Overexpression of sgr gene activates the loss of chlorophyll and induces early senescence in the

developing leaves (Park et al., 2007). Sgr synthesis has been reported in many plants such as

sorghum (Tao et al., 2000), maize (Tollenaar and Daynard, 1978; Crafts-Brandner et al., 1984a,

b; Bekavae et al., 1995; Rajcan and Tollenaar, 1999), rice (Buchanan-Wollaston, 1997; Cha et

al., 2002; Park et al., 2007), durum wheat (Spano et al., 2003), tomato (Akhtar et al., 1999;

Barry et al., 2008), pea (Sato et al., 2007) A. thaliana (Oh et al., 2000; Ren et al., 2007), pepper

(Barry et al., 2008), oat (Helsel and Frey, 1978) and Festuca pratensis (Armstead et al., 2006).

Stay-green regulates the loss of chlorophyll by disassembly of light-harvesting

chlorophyll-binding protein (LHCP) during senescence (Park et al., 2007). The stay-green is

characteristics of delayed senescence in mutants of durum wheat and increases 10-12% grain

weight (Spano et al., 2003). Tollenaar and Daynard (1978) demonstrated that some maize

varieties such as L087602 shows stay green phenotype, which increases the water, carbohydrates

and protein contents in the husks, cobs and seeds. FS854 is non-irrigating stay green maize

variety, which remained green after ear removal. It decreased more nitrogen and nitrate reductase

activity as well as chlorophyll and carboxylase enzymes (Crafts-Brandner et al., 1984b).

Nguyen (1999) demonstrated that stay-green genes delay leaf senescence in sorghum,

help the sorghum to cheat the heat and are involved in normal grain filling and reduce lodging in

heat stress and low moisture areas. In fact stay-green is used as a selection criterion in warm

areas (Acevedo et al., 1991, Kohli et al., 1991). For instance, most lines of wheat are sensitive to

heat stress and some lines are heat tolerant due to stay green (Rehman et al., 2009).

2.4 Heat tolerance in maize

Maize (Zea mays L.) is third important cereal after wheat and rice in Pakistan. It is extensively

grown in temperate, subtropical and tropical regions of the world. Maize shows optimum growth

at 28-31oC (Medany et al., 2007). High temperature is a constraint for maize growth. High

temperature decreased the shoot dry mass, RGR and NAR in maize significantly (Ashraf and

Hafeez, 2004). High temperature caused a distinct decrease in growth and photosynthetic

parameters (Karim et al., 2000), while chlorophyll fluorescence showed close association with

26

heat tolerance in maize (Lafitte and Edmeades, 1997). It caused wilting, top firing of leaves,

tassel blast (Smith, 1996), pollen abortion (Smith, 1996), poor grain set (Johnson and Herrero,

1981) and reduces the yield in maize (Herrero and Johnson, 1980; Maestri et al., 2002). The

kernel dry weight reduced to 79-95% in field conditions in B-73 inbred line of maize under high

temperature stress (Commuri and Jones, 2001).

Several major tolerance mechanisms, which are significant to offset heat stress include

free radical scavenging, ion transport, osmoprotection, synthesis of LEA proteins and factors

linked to signal transduction and transpirational control (Wang and Luthe, 2003). Heat stress

induced the synthesis of constitutive polypeptides at 40oC in maize (Law et al., 2001; Law and

Craft-Brandner, 2001), which prevented the cells from injury of abiotic stress including high

temperature (Noctor et al., 1998). In maize, high temperature induced the synthesis and

chloroplast protein synthesis elongation factor EF-TU, which defended the chloroplasts proteins

from heat-induced damage (Ristic et al., 2004; Momcilovic and Ristic, 2004). Higher membrane

stability and reduced generation of ROS are taken as important criteria of stress tolerance under

heat stress (Bravo and Uribe, 1981; Wahid et al., 2008).

Extreme temperature may prove lethal for plants grown in natural environment in the

absence of quick acclimation response. In these circumstances, there is a greater significance of

acquired thermo-tolerance. High temperature induced the oxidative stress and plants have

antioxidant defense systems against high temperature which is correlated with acquired thermo-

tolerance (Maestri et al., 2002). For instance, Thermo-tolerance in wheat was associated with

activity of CAT, SOD, ascorbate peroxidase and glutathione reductase in response to extreme

temperature (Sairam and Tyagi, 2004). A high uptake of Ca2+ and some calmodulin related genes

increased the activity of antioxidant enzymes during extreme temperature (Gong et al., 1997a)

and induced thermo-tolerance in seedling of maize (Gong et al., 1997b).

Elevated temperature induces numerous genes encoding transcriptional factors, which are

involved in heat stress tolerance (Chen and Zhu, 2004; Kotak et al., 2007). Different molecules and

ions play role in signal transduction and temperature sensing. Many genes encoding proteins such as

MAPK kinase, CDPK kinase, SOS3, Ca2+ sensor family and also various transcription factors. These

are involved in the signaling pathways, which are regulated under various abiotic stresses including

environmental temperature (Ichimura et al., 2000). Cytosolic Ca+2 sharply rise in response to

extreme temperature and involved in the acquired thermo-tolerance in plants (Larkindale and

27

Knight, 2002; Sung et al., 2003). High temperature increases the amount of K+ in maize which acts

as an osmolytes and improves cell water balance under a variety of environmental stresses including

heat stress (Bravo and Uribe, 1981). The stability of mRNA, translation efficiency and changes in

protein activity increased in plants including maize as a result slowing of gene expression (Sullivan

and Green, 1993).

The regulation of gene expression and stress tolerance mechanisms play important roles in

response to heat-shock. The synthesis and fast accumulation of HSPs could play a key role in the

protection of the metabolic apparatus of the cell. The accumulation and synthesis of HSPs through

HSFs network play a significant role in maize heat tolerance (Kuzmin et al., 2004). Heat tolerance of

maize was improved by moderate heat shock or gradually increased temperature (Gong et al., 2001),

which was associated with the synthesis of a new activase polypeptide (Crafts-Brandner and

Salvucci, 2002). These findings showed the expression of such genes in response to heat stress

appeared to play a great role in heat tolerance.

28

CHAPTER-3 MATERIALS AND METHODS

Maize is a multipurpose commercial crop and is cultivated extensively throughout Pakistan in

two seasons. Although it is a warm-season crop, it shows great variation in growth and

productivity if the ambient temperature is supra-optimal. Rapid release of greenhouse gases in

the atmosphere is not only continuously increasing global warming but also polluting the

environment. So it was deemed dire appropriate to study the genotypic differences, and growth,

physiological and molecular responses of maize to increasing ambient temperature when grown

in a glasshouse like environment. For this purpose, major part of work was completed in

Faisalabad, Pakistan and remaining part in Sendai, Japan.

EXPERIMENTS IN FAISALABAD, PAKISTAN

To select the maize varieties for the determination of mechanism of tolerance to glasshouse

conditions, preliminary experiments were conducted on five high yielding maize varieties. These

varieties were screened across two seasons in two years; winter and summer of 2005 and 2006.

The details on the methodology used to conduct these experiments have been presented below.

3.1.1 Source of maize seed, treatment and plant growth conditions

For screening purpose, seeds of five maize varieties (Sadaf, Agatti-2002, Agatti-85, SWL-2002

and EV-5098) were obtained from Maize and Millets Research Institute (MMRI), Yousafwala,

Sahiwal, Pakistan. The experiments were conducted in the wirehouse of the Department of

Botany, University of Agriculture Faisalabad, Pakistan. Seeds (12 in number) of all the varieties

were grown in plastic pots (dimensions 30 cm high, circumference of 82 cm at top and 70 cm at

bottom). A hole was made in the bottom for leaching during replacement of the soil solution.

Each pot contained 13 kg of dry sand, which was thoroughly washed with tap water followed by

distilled water before filling. All the pots were applied with 2 L of the half strength nutrient

solution (Hoagland and Arnon, 1950; Table 1), which was replaced after every four days. The

design of the experiment was completely randomized (CRD) with four replications per

29

treatment. After germination five uniform and healthy looking seedlings were retained for

making determinations at seedling, silking and grain filling stages.

3.1.2 Glasshouse (high temperature) treatment

Two open door plexiglass canopies 2 m high, 1.5 m long and 1.5 m wide were prepared by using

stainless steel frame and silicone rubbers (Fig. 1). Glasshouse conditions were created by shifting

the pots containing growing plants to the canopies at seedling, silking and grain filling stages,

while the control set was kept outside the canopies. For these pots, the roof of net house was

covered with polythene sheet to produce the shading effect like canopy. The temperatures and

RH inside and outside the canopies was recorded regularly during various times of the day/night

throughout the experimental period (Fig. 2). The data were recorded for growth and yield

attributes at seedling, silking and grain filling stages, and yield attributes at maturity. The plants

were kept inside the canopies for 15 days at the individual growth stages, and then harvested.

3.1.3 Growth and yield determination

Leaf area was taken of the plants as maximum leaf length × maximum leaf width × 0.68

(correction factor worked out for all leaves). Shoot length was taken of the pot grown plants

while root length was taken after carefully removing the roots from sand. Fresh weights of both

Table 1: Composition of nutrient solution (Hoagland and Arnon, 1950)

Constituents Concentration Macronutrients

Calcium nitrate 3.54 mM Potassium nitrate 5.00 mM Magnesium sulphate 2.00 mM Potassium dihydrogen phosphate 1.02 mM

Micronutrients

Boric acid 92.2 µM Manganese sulfate 2.19 µM Zinc chloride 1.62 µM Copper sulfate 0.69 µM Sodium molybdate 0.29 µM Na-Fe-EDTA 0.15 µM

All the salts were dissolved and mixed after autoclaving separately. Final pH of the solution was adjusted at 6.7.

30

Fig. 1: Plexiglass-fitted canopies used to create high temperature environment for maize. Light transmission index of the canopy was 75-80%.

Winter-2007

0

10

20

30

40

50

Mar-07 Apr-07 May-07 Jun-07

Outside canopyInside canopy

0

10

20

30

40

50

60

70

Mar-07 Apr-07 May-07 Jun-07

Outside canopyInside canopy

Summer-2007

0

10

20

30

40

50

Aug-07 Sep-07 Oct-07 Nov-07

Outside canopyInside canopy

01020304050607080

Aug-07 Sep-07 Oct-07 Nov-07

Outside canopyInside canopy

Temperature (oC) Relative humidity (%)

Fig. 2: Variation in the temperature and relative humidity inside and outside the plexiglass fitted canopy during experiment in Winter and Summer seasons in 2007

31

shoot and root were taken immediately after harvesting. For dry weights, the shoot and roots

were put in the paper bags and kept in an oven at 70oC for a week. For cob characteristics and

grain yield, the cobs were removed at maturity. The number of rows per cob and number of

grains per row were counted. The grains were extracted from the cobs and their yield was

assessed after weighing to express on per plant basis. The harvest index (HI) was calculated as:

HI (%) = (grain yield per plant) x 100/ (straw yield per plant)

The screening experiment led to the selection of two varieties; a heat tolerant (Sadaf) and

heat sensitive (Agatti-2002), on the basis of growth and yield characteristics at respective growth

stages in the above mentioned seasons.

3.2 Mechanism of heat tolerance in maize across the winter and summer seasons

The detailed mechanism of heat tolerance was studied at seedling, sikling and grain filling stages

of maize varieties Sadaf (tolerant) and Agatti 2002 (sensitive), selected from the screening

experiment.

3.2.1 Growth and yield determinations

The induction of treatments at all growth stages and growth measurements at seedling and

silking and grain yield characteristics at maturity were the same as given above. The design of

growth and yield experiments was completely randomized factorial with four replications.

3.2.2 Leaf water and osmotic relations

Third fully expanded leaf from top was excised to determine the leaf water potential (ψw) using

pressure chamber (Scholander Pressure Bomb, Arimad 2, Germany). The data was recorded

from 10:00 to 11:00 am. To determine the osmotic potential (ψs), the leaf from the same position

was excised, quickly frozen and kept in a freezer at -30oC. After about seven days, these leaf

samples were thawed at room temperature, put in a plastic syringe and sap expressed by applying

pressure and collected in a microfuge tube. The ψs was determined using osmometer (vapor

pressure based, Model-Wescor 5520, Utah). Turgor potential (ψp) was determined as difference

between the water potentials and osmotic potentials values as:

Ψp = ψw – ψs

32

For the determination of relative water contents (RWC), 10 leaf discs (1 cm square) cut

using cork-borer from third leaf from the top and weighed immediately for fresh weight (FW).

The discs were floated on water for overnight, weighed again next morning to obtained turgid

weight (TW). Dry weights (DW) were measured having dried the discs at 70oC in an oven for

one week. The RWC was calculated as given by Turner (1981):

% RWC = (FW-DW) × 100 / (TW-DW).

Total free amino acids were determined according to Hamilton and Van-Slyke (1943).

For this purpose, 1 mL of the aqueous extract of the sample was taken in test tube (25 mL

volume) and added 1 mL each of 10% aqueous pyridine solution and 2% ninhydrine solution

(prepared by dissolving 2 g ninhydrin in 100 mL distilled water). Then heated these test tubes in

boiling water in a water bath for about 30 min and transferred the solution to 50 mL test tube and

made the volume in each tube to 50 ml with distilled water. The absorbance of the colored

solution was taken at 570 nm using spectrophotometer. A standard curve was developed with

Lucine and calculated the free amino acids by the formula given below.

Total amino acids (mg/g Fresh weight) =

Graph reading of samples ×Volume of the sample × dilution factor

Weight of the tissue ×1000

Free proline was spectrophotometrically determined using the protocol of Bates et al.

(1973). Third leaf from the top (0.5 g) was homogenized in 5 mL of 3% of aqueous

sulphosalicylic acid and homogenate filtered through Whatman No.2 filter paper. One mL of

filtrate was taken and mixed with 1 mL of acid ninhydrin (1.25 g ninhydrine in 30 mL glacial

acetic acid) and 1 mL of glacial acetic acid in a test tube. The mixture was briefly vortexed and

heated at 100oC in a water bath for 1 h and then terminated the reaction in the ice bath. Four mL

of toluene was added to the solution and vortexed for 15-20 seconds while cool. The

chromophore containing proline was aspirated from aqueous phase in a test tube and warmed to

laboratory temperature. The absorbance was taken at 520 nm using spectrophotometer. The same

procedure was followed for blank using 2 mL of toluene. Standard curve was constructed using

proline (10 to 50 µg/2 mL). The amount of free proline was calculated by following formula:

33

Proline (µmoles)/ g fresh weight = [(µg proline/mL ×mL toluene)]/ [115.5 µg/umole]/ [(g sample/5]

3.2.3 Membrane characteristics and oxidative stress

The membrane permeability was expressed in terms of ion leakage from the leaves under stress.

Fresh third leaf tissue (0.5 g) was taken, put in 10 mL distilled water, vortexed for 5 sec and

measured for electrical conductivity at 0 h (EC0). The test tubes containing leaves in distilled

water were covered with aluminum foil and placed in refrigerator at 4oC for 24 h and measured

for electrical conductivity (EC1). Then these test tubes were autoclaved and electrical

conductivity of dead tissues (EC2) of the filtrate was measured. The relative membrane

permeability (RMP) was determined by applying the following formula of Yang et al. (1996):

RMP (%) = [EC1- EC0 / (EC 2 – EC 0)] ×100.

The H2O2 concentration was measured as described by Velikova et al. (2000). Third leaf

tissue (0.1 g) was homogenized in an ice bath with 1 mL of 0.1% (w/v) trichloroacetic acid

(TCA). The extract was centrifuged at 12,000×g for 15 min and 0.5 mL supernatant was added to

0.5 mL 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M potassium iodide. The

supernatant was vortexed and absorbance was read at 390 nm using water as blank. The H2O2

concentration was determined from standard curve prepared by using 35% H2O2.

The malondialdehyde (MDA) contents were determined as described by Heath and

Packer (1968). Fresh top third leaf tissue (0.1 g) was homogenized in 1 mL of 5% TCA and

centrifuged at 12,000×g for 15 min. Then 1 ml supernatant was mixed with an equal volume of

thiobarbituric acid (TBA) 0.5% in 20% (w/v) TCA. The mixture was boiled for 30 min at 95oC,

was cooled and centrifuged at 7500×g for 5 min to clarify the solution. The absorbance of the

mixture was recorded at 532 nm and 600 nm, while using 5% TCA as blank. Non-specific

turbidity was corrected by subtracting the absorbance at 600 nm from that taken at 532 nm.

MDA contents were calculated using its absorption coefficient of 155,000 nmol mol-1 as:

MDA (nmol mL-1) = [(A532-A 600)/155000]106

3.2.4 Leaf gas exchange and pigment analysis

Gas exchange characteristics were measured by an open system potable infrared gas analyzer

(IRGA; LCA-4, ADC, Hoddesdon, England) of third leaf (from top) of each plant. Measurement

34

were taken between 10.00 and 11.00 am with the leaf chamber adjustments: leaf surface area

11.35 cm2, ambient CO2 concentration 357 µmol mol-1, temperature of leaf chamber was at 32.5

− 37oC, leaf chamber gas flow rate (v) 392.8 mL/min, molar flow of air per unit leaf area 440

µmol m-2 s-1, ambient pressure (P) 99.6 kPa , water vapor pressure in to chamber ranged from

20.5 to 23.1 mbar, PAR (Q leaf) on leaf surface ranged from 975 -1250 µmol m-2 s-1

For the determination of chlorophyll a, b, their total and total carotenoids contents as

described by Arnon (1949), 0.5 g of the fresh third leaf from the top was homogenized with

pestle and mortar in 80% acetone and made the volume up to 5 mL and filtered. The absorbance

of the filtrate was read at 480 nm for the carotenoids, and at 645 and 663 nm for chlorophyll a

and b respectively using a spectrophotometer (Hitachi-U-2001, Japan). Chlorophyll a, b, their

total and ratio were calculated as describe by Yoshida et al. (1976), while total carotenoids were

computed with the formula of Daveis (1976), as given below:

Chl. a (mg/g) = [12.7(OD663)-2.69(OD645)] x V/1000 x W

Chl. b (mg/g) = [22.9(OD645) - 4.68(OD663)] x V/1000 x W

Total Chl. (mg/g) = [20.2(OD645) + 8.02 (OD663)] x V/1000 x W

Carotenoid (g ml-1) = {A Car / Em×100} Where: Em ×100 =2500,

A Car = [(OD 480) +0.114 (OD 663) – 0.638 (OD 645)]/2500

Where

V = Volume of the acetone used in extract (ml)

W = Weight of fresh leaf tissue (g)

3.2.5 Determination of ion and nutrients

The analysis of K+, Ca2+ and Mg2+ was done by the methods described by Yoshida et al. (1976).

The oven dried shoot and root were grinded in grinding mill. To determine different cations and

anions two types of digestions were done. The dried ground material (0.15 g) was placed in a test

tube followed by the addition of 3 mL of concentrated H2SO4 and incubated over night at room

temperature. Next morning, 1−2 mL of 35% AR grade H2O2 was poured down along the

sidewall of the test tube and rotated them, waited for some time for the reaction to occur, placed

them on a hot plate in a fume hood and temperature was raised gradually up to 350oC until fumes

were produced and continued heating for another 30 min. Removed the tubes from the hotplate

and cooled and slowly added 1 mL of H2O2 and repeated the above steps; the cooled material

was colorless. The final volume of sample of the extract was made up to 50 mL with distilled

35

water after filtration. The samples were preserved in a freezer until analyzed. The quantities of

K+, Mg2+ and Ca2+ were measured from this extract.

The analysis of K+ was carried out with flame photometer (Sherwood Model 410,

Cambridge). Standard curve of K was constructed by diluting the 1000 mg L-1 stock solution that

was prepared by dissolving 1.907 g of analytical grade KCl in deionized water and volume made

up to one liter. Ten mL of this solution was diluted to 100 mL to get 100 ppm K+. A graded

series of standards (ranging from 5 to 50 ppm) K+ were prepared and standard curves were

drawn. The values of unknown K+ samples were determined by comparing with standard curves.

Ca2+ and Mg2+ were determined by atomic absorption Spectrophotometer (Spectra AA-5,

Varian Company) using a fuel rich air-acetylene flame; 10 cm burner head, 0.7 nm slit width and

10 mA lamp current. Ca+2 and Mg+2 were measured at 422.7 nm, 285.2nm respectively. For

Ca2+, a graded series of standards ranging from 10 ppm to 100 ppm was made from stock

solution prepared by dissolving 2.247 g CaCO3 in 10 ml HCl, made up to 1 liter with distilled

water, giving a concentration of 1000 ppm Ca. For Mg2+, stock solution was made by dissolving

1 g magnesium ribbon in a minimum volume of (1+1) HCl by heating, diluted to 1 liter with

1%(v/v) HCl, which gave a concentration of 1000 ppm. Ten mL of this solution was diluted to

100 mL to get 100 mg L-1 Mg. Final quantities were computed by comparison of the sample

readings with the standard curves.

For the determination of soluble phosphates, dried ground material (0.15 g) was taken in

test tubes containing 10 mL of deionized water and boiled for 1 h, filtered and made the volume

up to 50 mL with distilled water. The extract (2.5 mL) was dissolved in 2.5 mL of Barton reagent

(prepared as described by Yoshida et al., 1976) and total volume was made as 25 mL. The

samples were vortex and kept for 20 min at room temperature. Measured the color intensity of

samples at 420 nm with spectrophotometer using water as blank and values of phosphate were

calculated using standard curve. A graded series of standards ranging from 2.5 to 15 mg L-1 of

PO4-2 was prepared to determine the exact quantity of phosphate in the unknown samples.

Soluble nitrates were determined as described by Kowalenko and Lowe (1973). The dried

ground material (0.5 g) was taken in test tubes containing 5 mL of deionized water and boiled for

1 h, filtered and made the volume up to 50 mL with distilled water. Three mL of the extract was

dissolved in 7 mL of working CTA solution with the thrust of a pipette filler and vortexed

briefly. Stock solution of NO3-1 (100 mg L-1) was prepared by dissolving 0.7216 g of pure dried

36

KNO3 in a liter of distilled water. A graded series of standards ranging from 10 to 100 mg L-1 of

NO3-1 was prepared by dilution of the stock solution. After 20 min, the intensity pf the yellow

colored complex so formed was taken at 430 nm on a spectrophotometer. The water was used as

a blank and values of soluble nitrate were calculated using standard curve.

EXPERIMENTS AT TOHOKU UNIVERSITY, SENDAI, JAPAN

3.4 Gene expression in maize under heat stress

Plants show the varied expression of many genes, which have close relevance to plant stress

tolerance. In this part of the thesis, determination for the expression pattern of four genes was

made; dehydrin2 (dhn2), heat shock protein70 gene (hsp70), senescence associated gene (sag)

and stay green2 (sgr2) gene. The protocols for these determinations are given below.

3.4.1 Plant materials and experimental detail

Seeds of yellow popcorn (Zea mays L.) were donated by Mr. Hideo Tokairin to Genomic

Reproductive Biology Lab, Tohoku University Japan. Seeds were sown in Petri dishes (9 cm in

diameter) lined with three sheets of filter paper and wetted with 12 ml 0.2% plant preservative

material solution (PPM). The petri dishes were incubated at 30oC, in the darkness at 50% RH.

After 4 days, plants were shifted to Leonard Jar having three layers of filtered papers. For

control, filter paper were fully wetted with distilled water and completely covered the jar while

for heat stress, plants were shifted to NK system Biotran (Nippon, Medical and Chemical

Instrument Co, LTD, Japan) in the graduate School of Life Sciences, Tohoku University.

Temperatures were 28/22oC, 42/36oC (day/night) for control and heat stress respectively and

light and dark cycle of 14/10 h. The samples were harvested after 1h, 3h, 6h, 24h, 48h and 72h,

respectively. Leaf samples were frozen with liquid N and stored at -80oC until used. Design of

experiment was completely randomized (CRD) with three replications per treatment.

3.4.2 RNA extraction and quantification

Plant material (0.1 g) was homogenized in 1 mL of TRIzol reagent (Invitrogen, CA), and

incubated for 5 min at 25oC. The tubes were processed in the Fast RNA (R) Pro Green Kit (Fast

Prep ® instrument) for four times and 10 s at setting of 6.0, and the rest period between each

37

time was 1 min. Samples were added with 0.2 mL of chloroform in each sample containing 1 ml

of TRIzol Reagent, vortexed vigorously for 15 s, centrifuged at 12000 rpm for 15 min at 4oC and

kept 2-3 min for rest of the period. The mixture was separated after centrifugation into a

colorless upper aqueous phase, an interphase and a lower red phenol-chloroform phase.

For RNA precipitation, 0.4 mL of aqueous phase (without disturbing the interphase) was

transferred to a new microcentrifuge tube and then added 0.4 mL of isopropanol in each tube,

kept for 10-30 min at room temperature and then centrifuged at 12000×g for 10 min at 4oC. The

RNA precipitation occurred like pellet on the side and bottom of the tube; discarded the

supernatant and washed the RNA pellet with 1 mL of 70% ethanol, quickly vortexed (for 10 sec)

and centrifuged again at 12000×g for 10 min at 4oC. After isolation, total RNA was quantified by

taking absorbance at 260 nm using DEPC treated water as a blank. The RNA samples were

stored at -70oC.

3.4.3 Agarose gel electrophoresis

RNA integrity was determined by analyzing a portion of the RNA sample using 1.8% agarose

gel electrophoresis. Forty ml of Tris Buffer and 0.72 g Agarose was added in a screw capped

bottle and placed these bottle in to microwave oven for boiling, removed temperature was about

70oC, then added 2 µL of (10 mg mL-1) ethedium bromide (final concentration of 0.5 µg mL-1).

The gel was poured, when polymerized, filled the tank with running buffer TBE (0.045M tris-

borate and 0.001 M EDTA) containing 20 µL ethidium bromides in the running buffer. RNA

samples were loaded into the wells along with Phi*174HaeIII (1 µl sample+ 5 µl loading dye;

Bromo phenol blue+glycerol+buffer TBE), which was used as marker. The samples were

electrophoresed under electric field at 100 V for 30 min.

3.4.4 Synthesis of cDNA for RT-PCR

The reverse transcription polymerase chain reaction (RT-PCR) was performed with 2 µL of each

RNA sample and the PrimeScript®RT reagent Kit (TaKaRa. Bio Inc. Japan). The protocol for

cDNA synthesis has been given as in Table 2.

Eight µL of reaction mixture was mixed with 2 µL sample in a microfuge tube by

pipetting and placed on the ice to carry out the reaction at the same time. The cDNA synthesis

reaction was incubated in PCR (THERMAL CYCLER PERSONAL TAKARA. Co. Japan) at

38

37oC for 15 min followed by 85oC for 5 s, and was then cooled to 4oC. The mixture was then

diluted by the addition of 30 µL of easy dilution solution (TaKaRa. Bio Inc. Japan, code

no.9160) after the RT.

3.4.5 Primer designing

Primers were designed for different genes. Forward and reverse primers were selected from Exon

I and Exon II of DNA database, respectively. Gene-Specific RT-PCR primers for genes (both

reference and target) were designed to conserved regions on the basis of comparison of

sequences from rice and maize ESTs (Iskandar et al., 2004). Sequences for maize were obtained

from National Center for Biotechnology information (http://www.ncbi.nlm.nih.gov/). Primers

were prepared with software available at http://www.riken.go.jp/lab-www/help2/members/

kagawa/tmnumber.html to set to an annealing temperature (58-60oC). All primers were

synthesized commercially by Exigen Co. Ltd., Japan (Table 3). The homology of gene sequences

to maize ESTs of each target gene was examined by a BLASTn search of the ESTs confined to

Japonica rice cultivar. The complete nucleotide sequences of a maize elongation factor1 Alpha

(EF1α), dehydrin (dhn2), heat shock protein70gene (hsp70), senescence associated gene (sag)

and stay green 2 (sgr2) were reported with accession numbers (Table 3).

To measure the expression difference in each gene, RT-PCR was performed by

MiniOpticon (Bio-Rad, Hercules, CA) with SYBR Prime ScriptTM RT-PCR Kit (TaKaRa Bio

Inc. Japan). The protocol for the quantitative RT-PCR has been given as in Table 4. During the

real-time PCR step, Q-solution (Qaigen Co. Ltd, USA) was used to improve amplification of

cDNA templates with high GC contents. The reaction was carried out in the PCR Detector

System (Chromo 4, Real time PCR System, Bio-Rad) with the conditions: denaturation at 95oC

Table 2: The protocol for cDNA synthesis Reagent Amount Final Concentration 5* Reaction buffer 2.00 µL 2*14 ul DEPC.H2O = 28 µL (Prime script Buffer) #1 30.00 µL Prime Script RT Enzyme # 2 0.5 µL 0.5*3.5µl DEPC.H2O = 7 µL Oligo dt Primer (50 uM) # 3 0.5 µL 0.5*3.5 µl DEPC.H2O l = 7 µL Random 6mer (100uM) # 4 0.5 µL 0.5*3.5 µl DEPC.H2O = 7 µL Amount of RNA sample 2 µL 2 µL RNase free water # 5 4.5 µL 4.5*14 µl DEPC.H2O = 63 µL Total amount of Reagent 10 µL

39

for 30 sec, followed by 40 cycles of 95oC for 5 sec, 60oC for 20 sec and then a final extension at

72oC for 10 sec. After the completion of reaction, verified the amplification curve and melting

curve, and Ct value was calculated from the amplification curve. EF-1α was used as an internal

standard to normalize for the variation in the amount of cDNA template. Transcript level of the

specific gene at a different time with different treatment was firstly normalized to a standard

(EF1α) using the formula.

ΔCt (specific gene) = Ct (EF1α) - Ct (specific gene).

The relative mRNA level was calculated with the formula:

Transcript level= 2^ΔCt (specific gene)/ 2^ΔCt (controls).

RT-qPCR experiments were performed in triplicate for each sample. The PCR products

were resolved by electrophoresis in 1.2% agarose gel, stained with ethidium bromide and

visualized under ultraviolet light.

3.5 Statistical analysis

All the determinations were made in quadruplicate from this completely randomized experiment.

The presence or absence of significant differences among different factors was ascertained with

analysis of variance (ANOVA). Computer software COSTAT (CoHort software, 2003,

Table 3: Gene specific PCR primers for quantitative RT-PCR amplification for maize

Name of gene

Accession No. Amplicon size (bp)

Primer Pair Sequence

EF1α NM_00111149 160 F: gct ggt atc tcc aag gac ggc R: ggt agg atg aga ctt cct tca caa tct c

dhn2 L35913 157 F: gcc gag aag aag gac agc ctt c R: ggt ggt ggt cgc cct cat c

hsp70 pMON9502 510 F: cgc acc acg ccg tcc tat gt R: cgt gcc acc acc aag gtc g

sag EU967002 109 F: caa gag cct gca gga caa gaa c R: gta ggt gta gcc gca gct ggt

sgr2 AY850139 132 F: tcc agc tcc ggg tgc c R: ctc cgg cgg cca ctt c

40

Monterey, California) was used for all statistical analysis and MS-Excel was used to graphically

present the data.

3.6 Chemicals

All chemicals used in the experiments were purchased either from Biorad, Sigma, Merck, BDH,

Fluka and Aldrich, and were of either ACS or AR grade.

41

CHAPTER-IV RESULTS AND DISCUSSION

Global warming is leading to the crippled crop productivity and is changing the cropping around

the globe. Heat stress is posing as a great problem during winter and summer seasons. The

emerging threats due to heat stress warrant comprehensive understanding the crops responses to

it. Keeping in view the importance of global warming as a threat to crop production, studies were

initiated on maize to understand some basic physiological and molecular phenomena in two

selected differentially heat tolerant varieties in winter and summer seasons under glasshouse

conditions. It is worth mentioning that most part of the studies was performed at University of

Agriculture, Faisalabad, Pakistan, while some part was completed in Tohoku University, Sendai,

Japan. Summary of the results on the experiments are narrated below:

STUDIES AT FAISALABAD, PAKISTAN

The studies at Faisalabad, Pakistan comprised selection of varieties after preliminary screening

from a lot of locally available varieties of maize. These selected varieties were subjected to

extensive experimental trial at three growth stage over two seasons in normal (control) and

glasshouse conditions. The determinations were made for the alterations in growth and yield

characteristics, photosynthetic responses, photosynthetic pigments, water and osmotic relations,

membrane characteristics, and nutrition relationships. Details of these experimental trials are

elaborated below.

42

4.1. Growth and yield characteristics

4.1.1 Results

a. Shoot length

At seedling stage, during winter season heat treatments, while during summer season varieties

indicated significant difference, while there was no interaction of varieties and treatments during

these seasons. In winter season, shoot length was similar in both the varieties under control

condition, while under glasshouse condition, Sadaf performed better than Agatti-2002. However,

in summer season Sadaf showed greater shoot length than Agatti-2002 in both the conditions.

Plants grown in winter were much shorter than those grown in the summer season (Fig. 3).

Shoo

t len

gth

(cm

)

0

40

80

120

160

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

ControlGlasshouse

b

c

a

c

0

40

80

120

160

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

0

40

80

120

160

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 11.11ns 106.77ns Treatments (T) 1 117.38** 164.70* V × T 1 2.78ns 1.78ns Error 12 7.34 27.67

Varieties (V) 1 6240.76*** 100.56* Treatments (T) 1 225.08ns 507.50** V × T 1 793.46** 1.30ns Error 12 49.54 14.65

Varieties (V) 1 21.11ns 126.77ns Treatments (T) 1 147.38** 104.70* V × T 1 12.78ns 21.78ns Error 12 7.34 27.67

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons

Fig. 3: Changes in shoot length of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages. In this and subsequent figures, vertical lines on the bars are standard deviation of means

43

At silking stage, in winter season, data revealed significant difference in the varieties, but

a non-significant difference in the treatments with a significant interaction of these factors.

However, in summer a significant difference was noted in the varieties and treatments, but there

was no interaction of these factors. In winter, under glasshouse condition, Sadaf showed greater

shoot length than Agatti-2002 under both the conditions. Shoot length in the glasshouse grown

plants of Sadaf was higher, while a reverse trend was noted in Agatti-2002. However, in summer

season under glasshouse, Sadaf and Agatti-2002 showed greater shoot length than in control

condition. Plants grown in winter were longer than those in summer (Fig. 3).

At grain filling stage, statistical analysis of data revealed non-significant difference in the

varieties, a significant difference in the treatments but a non-significant interaction of these

factors in both the seasons. In both seasons, Sadaf performed better than Agatti-2002 both under

control and glasshouse conditions. In both the seasons, shoot length was greater under heat stress

than control in Sadaf, while in glasshouse grown plants of Agatti-2002, the value of this attribute

was lesser than control in both the seasons (Fig. 3).

b. Root length

At seedling stage, in winter season, there was no significant difference in the varieties, but a

significant difference was evident in the treatments and a no significant interaction of these

factors. However, in summer season, a significant difference was noted in the varieties but non-

significant one in the treatments, and a non-significant interaction of both the factors for root

length. During winter season, both the varieties indicated a similar pattern of changes in root

length. However, glasshouse grown plants displayed lesser root length as compared to respective

controls in both varieties. In summer season, although root length was greater in Sadaf, both the

varieties showed relatively increased root length than the corresponding controls (Fig. 4).

At silking stage, statistical analysis of data revealed significant differences in the varieties

and treatment, and with significant interaction of these factors in winter season. However, in

summer season, no significant difference in the varieties but a significant one in the treatments,

and a non-significant interaction of both the factors was noted. In winter season, Sadaf exhibited

longer roots than control, but reverse behavior was noted in Agatti-2002 under glasshouse

condition. Although much shorter than the winter season plants, glasshouse grown plants of both

44

the varieties in summer season indicated longer root length in both varieties as compared to

respective controls (Fig. 4).

At grain filling stage, statistical analysis of data revealed non-significant difference in the

varieties, while significant difference in treatments with non-significant interaction of these

factors during winter and summer seasons. In winter season, there was no difference in root

length of control and glasshouse grown plants, which decreased in latter condition in Agatti-

2002. Although much shorter than the winter season plants, control and glasshouse grown plants

of both the varieties in summer season indicated no specific difference in the root length (Fig. 4).

c. Shoot dry weight At seedling stage, in winter season, data showed significant difference

in the varieties and treatments with a significant interaction of both factors. However, in summer

Roo

t len

gth

(cm

) 0

10

20

30

40

50

60

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007Se

edlin

g

ControlGlasshouse

a a

a

b

0

10

20

30

40

50

60

Sadaf Agatti 2002 Sadaf Agatti 2002

Silk

ing

0

10

20

30

40

50

60

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 4.69ns 370.56** Treatments (T) 1 424.25** 16.67ns V × T 1 11.12ns 3.06ns Error 12 9.71 4.52

Varieties (V) 1 85.56** 9.50ns Treatments (T) 1 52.57* 101.68** V × T 1 88.68** 2.50ns Error 12 8.32 5.99

Varieties (V) 1 71.11ns 56.77ns Treatments (T) 1 97.38** 74.70* V × T 1 12.78ns 6.78ns Error 12 7.34 27.67

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 4: Changes in root length of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

45

season there was significant difference in the varieties and treatments but no interaction of both

factors was evident. In winter, although shoot dry weight was quite low, glasshouse condition

further reduced it in both the varieties. In summer season, shoot dry weight was not affected

remarkably by glasshouse condition. Although Sadaf indicated a higher shoot dry weight than

Agatti-2002, glasshouse grown plants of both the varieties indicated greater value of this

attribute than Agatti-2002 (Fig. 5).

At silking stage, in winter season, data revealed significant difference in the varieties, and

treatments, also there was a significant interaction of both factors. Contrarily, in summer season

the varieties, but not the treatments, differed significantly as well as there was no interaction of

varieties and treatments for shoot dry weight. In winter, both the varieties had similar shoot dry

weight under control, while under glasshouse condition Agatti-2002 manifested significantly

Shoo

t dry

wei

ght (

g/pl

ant)

a cb d0

12

24

36

48

60

72

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Control Glasshouse

ab ba

c

0

12

24

36

48

60

72

Sadaf Agatti 2002 Sadaf Agatti 2002

Silk

ing

Seasonsa

b baa

c

ab

c

0

12

24

36

48

60

72

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 5.73** 9.21** Treatments (T) 1 2.44** 1.66** V × T 1 0.64** 0.01ns Error 12 0.06 0.30

Varieties (V) 1 100.17** 12.15* Treatments (T) 1 23.14* 2.10ns V × T 1 44.22** 3.63ns Error 12 2.94 1.78

Varieties (V) 1 14592.64** 924.16ns Treatments (T) 1 1375.67ns 5574.12** V × T 1 2668.76* 7951.29** Error 12 422.28 13.33

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 5: Changes in shoot dry weight of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

46

reduced shoot dry weight than Sadaf. In summer season, although lesser than winter, both the

varieties had similar shoot dry weight under control, while under glasshouse condition it was

greater than control in Sadaf but equal to control in Agatti-2002 (Fig. 5).

At grain filling stage, in winter season, data revealed significant difference among the

varieties but not the treatments, while there was a significant interaction of both factors.

However, in summer season, the varieties differed non-significantly while treatments indicated

significant difference with a significant interaction of varieties and treatments. In winter season,

although Sadaf indicated a higher shoot dry weight under either condition, glasshouse condition

greatly reduced this attribute in Agatti-2002. In summer season, under control condition although

shoot dry weight was relatively lesser in Sadaf than Agatti-2002, glasshouse condition did not

influence this variable in Sadaf but decreased remarkably in Agatti-2002 (Fig. 5).

d. Root dry weight

At seedling stage, in winter season, statistical analysis of data revealed no significant difference

among the varieties but a significant one in the treatments, with no significant interaction of both

factors. However, in summer season, there was a significant difference in the varieties, but non-

significant difference in the treatments, as well as there was no interaction of both these factors.

In winter season, both the varieties indicated similar root dry weight under control but a reduced

one under glasshouse condition. In summer season, on the other hand, both the varieties showed

similar response both under control and glasshouse condition for this parameter (Fig. 6).

At silking stage, in both the seasons, there was significant difference in the varieties and

treatments with a significant interaction of both the factors for this attribute. In winter, root dry

weight was greater in Sadaf, which increased further under glasshouse condition, while in

Agatti-2002 glasshouse condition reduced it compared to control. In summer, the root dry weight

increased in Sadaf under glasshouse condition as compared to control, but in Agatti-2002 root

dry weight was similar under both the conditions (Fig. 6).

At grain filling stage in winter season, there was no significant difference in the varieties

and treatments but there was significant interaction of both factors. However, in summer season,

a significant difference was notable in the varieties, while non-significant difference was evident

in the treatments with a non-significant interaction of both the factors. In winter season, root dry

weight was much greater in Sadaf, which increased further under glasshouse condition, while in

47

Agatti-2002, a greatly reduced root dry weight was noted under glasshouse condition. In

summer, although root dry weight was higher in both the varieties as compared to winter season,

the trend of changes did not differ much from that observed under winter season (Fig. 6).

e. Number of leaves per plant

At seedling stage, statistical analysis of data recorded from winter season crop revealed

significant difference in the varieties and treatments but there was no significant interaction of

both factors. However, in summer season crop, only the varieties differed significantly while no

difference was evident in the treatments, as well as an interaction of varieties and treatments was

missing. In winter season crop under control condition, number of leaves per plant was relatively

lesser in Sadaf than Agattti-2002, while in glasshouse both these varieties showed similar value

Roo

t dry

wei

ght (

g/pl

ant)

0

4

8

12

16

Sadaf Agatti 2002 Sadaf Agatti 2002

Seed

ling

ControlGlasshouse

a b b ca

c

ad

0

4

8

12

16

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasonsa aa

b

0

4

8

12

16

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.235ns 0.378* Treatments (T) 1 0.971** 0.013ns V × T 1 0.000ns 0.013ns Error 12 0.402 0.064

Varieties (V) 1 6.30** 5.25** Treatments (T) 1 0.67* 1.19** V × T 1 1.99** 2.49** Error 12 0.12 0.09

Varieties (V) 1 0.0638ns 11.672** Treatments (T) 1 0.0625ns 1.174ns V × T 1 5.842* 3.061ns Error 12 0.845 1.063

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 6: Changes in root dry weight of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

48

of this number. In summer season, this number was greater than that observed in winter season,

while glasshouse condition did not influence this attribute in both the varieties (Fig. 7).

At silking stage, in winter season, statistical treatment of data revealed significant

difference in the varieties, non-significant difference in the treatments and a significant

interaction of both the factors. On the other hand, in summer season, the varieties, treatments and

interaction of these factors were non-significant. In winter, number of leaves was greater in

Sadaf than Agatti-2002, which increased in the former and decreased in the latter variety under

glasshouse condition. In summer season, both varieties under either condition showed no big

difference in this number (Fig. 7).

At grain filling stage in winter and summer seasons, data revealed non-significant

difference in the varieties, a significant one in the treatments while there was no interaction of

both these factors. In winter season, Sadaf displayed lower number of leaves per plant than

Num

ber

of le

aves

per

pla

nt

0

2

4

6

8

10

12

Sadaf Agatti 2002 Sadaf Agatti 2002

Seed

ling

ControlGlasshouse

a

b

a

b

0

2

4

6

8

10

12

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

0

2

4

6

8

10

12

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 4.69* 5.06* Treatments (T) 1 6.25* 0.34ns V × T 1 0.44 0.06ns Error 12 0.94 1.04

Varieties (V) 1 58.78** 0.028ns Treatments (T) 1 0.25ns 1.361ns V × T 1 4.34* 0.001ns Error 12 0.858 0.514

Varieties (V) 1 11.11ns 106.77ns Treatments (T) 1 117.38** 164.70* V × T 1 2.78ns 1.78ns Error 12 7.34 27.67

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 7: Changes in number of leaves per plant of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

49

Agatti-2002 under control condition. However, under glasshouse condition, Sadaf produced

leaves in greater number than Agatti-2002. In summer season on the other hand, this number was

greater in Sadaf than Agatti-2002 under control condition. Under glasshouse condition, Sadaf

showed an increased while Agatti-2002 a decreased number of leaves per plant (Fig. 7).

f. Leaf area per plant

As shown in Fig. 8, at seedling stage in winter season, statistical analysis of data revealed a

significant difference in the varieties and treatments but a non-significant interaction of both

factors. However, in summer season, the varieties, but not the treatments, indicated significant

difference, and no interaction of varieties and treatments was evident. In winter season, leaf area

Lea

f are

a (c

m2 /p

lant

)

0

40

80

120

160

200

Sadaf Agatti 2002 Sadaf Agatti 2002

Seed

ling Control

Glasshouse

bb

b b

a

ca

b

0

40

80

120

160

200

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

b c

bc

a

da

d

0

40

80

120

160

200

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 2474.29** 1431.31** Treatments (T) 1 402.45* 46.92ns V × T 1 28.24ns 0.56ns Error 12 60.88 21.96

Varieties (V) 1 8226.99** 98.29ns Treatments (T) 1 161.46ns 76.15ns V × T 1 3854.02** 171.50** Error 12 97.40 32.37

Varieties (V) 1 511.11** 106.77** Treatments (T) 1 1117.38** 164.70** V × T 1 52.78** 61.78** Error 12 7.34 7.67

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 8: Changes in leaf area per plant of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

50

per plant was lower in Sadaf than Agatti-2002 under control condition, while glasshouse

condition decreased it in both the varieties. In summer season, although Sadaf had greater leaf

area per plant, glasshouse effect did not affect this attribute much in both varieties.

At silking stage, data showed significant difference in the varieties, non-significant

difference in the treatments while a significant interaction of both the factors was notable. In

summer season, varieties and treatments indicated no significant differences while there was a

significant interaction of both these factors. In winter season, Sadaf with greater leaf area under

control indicated an increase in it under glasshouse condition. In summer season, leaf area per

plant was similar in both the varieties under control condition, while glasshouse condition

increased it in Sadaf but decreased in Agatti-2002 (Fig. 8).

At grain filling stage in both the seasons, data indicated significant differences in the

varieties and treatments with a significant interaction of both factors. In winter season, both the

varieties had similar leaf area under control condition, which increased in Sadaf but decreased in

Agatti-2002 under glasshouse condition. In summer season, although both the varieties displayed

lesser leaf area compared with winter crop, the trend of changes in this season was similar to that

observed in winter season (Fig. 8).

g. Cob characteristics

Data for the number of cobs per plant indicated no significant difference in the varieties but a

significant one in treatments, although there was no interaction of these factors. However, in

summer season, the varieties indicated significant but treatments showed non-significant

differences, and a significant interaction of these factors was evident. In winter season, Agatti-

2002 had relatively greater number of cobs per plant than Sadaf under control condition in both

seasons. However, under glasshouse condition in winter season, both the varieties indicated a

reduction, while in summer season Sadaf showed an increase but Agatti-2002 a decrease in the

number of cobs per plant (Fig. 9).

For number of rows per cob, data revealed non-significant difference in the varieties, a

significant one in the treatments while there was no significant interaction of these factors. In

summer season, the difference in the varieties, treatments and an interaction of these factors were

non-significant. In winter season, both the varieties indicated a greater number of rows per cob

51

than summer season under control condition. However, under glasshouse condition, reduction in

this number was much higher in winter than in summer season (Fig. 9).

The data regarding number of grains per cob indicated significant difference in the

varieties and treatments with a significant interaction of these factors in winter season. However,

bc aba

c

0.0

0.5

1.0

1.5

2.0

Sadaf Agatti 2002 Sadaf Agatti 2002

Num

ber o

f cob

s pe

r pl

ant Control

Glasshouse

0

4

8

12

16

Sadaf Agatti 2002 Sadaf Agatti 2002

Num

ber o

f row

s pe

r cob

0

30

60

90

120

Sadaf Agatti 2002 Sadaf Agatti 2002

Num

ber o

f gra

ins

per c

ob

0

50

100

150

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Fres

h w

eigh

t of c

ob (

g)

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.000ns 0.037* Treatments (T) 1 0.101** 0.000ns V × T 1 0.039ns 0.095** Error 12 0.009 0.006

Varieties (V) 1 0.25ns 2.83ns Treatments (T) 1 8.03** 4.62ns V × T 1 0.69ns 4.77ns Error 12 0.92 2.39

Varieties (V) 1 1167.37** 207.83* Treatments (T) 1 3080.22** 2154.47** V × T 1 793.34* 291.87ns Error 12 88.71 31.73

Varieties (V) 1 387.62* 75.00ns Treatments (T) 1 4432.22** 1347.93** V × T 1 79.44ns 87.17ns Error 12 56.51 64.34

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 9: Changes in some cob characteristics of control and glasshouse grown maize varieties during winter and summer seasons at grain filling stages

52

in summer season, varieties and treatments showed significant difference while there was no

interaction of these factors. In winter season under control both the varieties indicated a similar

number of grains per cob, which reduced substantially in Agatti-2002 under glasshouse

condition. In summer season, although the trend of changes was similar to the winter season, but

the reduction in this attribute under glasshouse condition were lesser than those noted in the

winter season (Fig. 9).

The cob weight indicated significant differences in the varieties and treatments with a

non-significant interaction of these factors. However, in summer season crop only treatments

indicated significant difference. The cob weight did not differ much in both the varieties and

seasons under control condition. In the glasshouse grown plants, although there was a reduction

in this attribute in both the seasons, a greater reduction was evident in winter season (Fig. 9).

h. Grain yield characteristics

Statistical analysis of data on grain yield per cob indicated significant differences in the varieties

and treatments in both the seasons but there was a significant interaction of both these factors in

winter season, while a non-significant interaction in summer season. Under control condition in

both the seasons, grain yield per cob was similar in both the varieties. However, under

glasshouse condition although this attribute decreased in both the varieties, Agatti-2002 indicated

a greater reduction, which was well explicit in the winter season (Fig. 10).

For 100 grain weight, results revealed that in winter season varieties and treatments,

while in summer season only treatments indicated significant differences, while there was no

interaction of these factors in both the seasons. In winter and summer seasons, although 100

grain weight was greater in Sadaf than Agatti-2002, glasshouse condition produced a greater

reduction in the latter variety (Fig. 10).

For grain yield per plant, data revealed significant difference in the varieties and

treatments with a significant interaction of both these factors in both the seasons. Under control

condition, grain yield per plant was relatively greater in winter than summer season. Under

glasshouse condition, although grain yield per plant reduced in both the varieties, Agatti-2002

indicated a greater reduction than Sadaf, and glasshouse condition was more adverse ti this

attribute in winter than summer season (Fig. 10).

53

For harvest index, results revealed that in winter season, only treatments while in summer

season varieties and treatments indicated significant differences, while there was no interaction

of these factors in both the seasons. In winter season, although harvest index was higher in

Agatti-2002 than Sadaf under control condition, glasshouse condition greatly affected this

parameter in the former variety. In summer season, Sadaf had greater harvest index than Agatti-

aa

b

c

0

20

40

60

80

100

Sadaf Agatti 2002 Sadaf Agatti 2002

Gra

in y

ield

per

cob

(g) Control

Glasshouse

0

20

40

60

80

Sadaf Agatti 2002 Sadaf Agatti 2002

100

grai

n w

eigh

t (g)

a aa a

b

c

a

b

0

20

40

60

80

100

Sadaf Agatti 2002 Sadaf Agatti 2002

Gra

in y

ield

per

pla

nt (

g)

0

10

20

30

40

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Har

vest

inde

x (%

)

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 559.50** 163.89** Treatments (T) 1 2301.96** 1194.53** V × T 1 145.59* 58.04ns Error 12 15.79 12.59

Varieties (V) 1 210.35** 9.63ns Treatments (T) 1 1259.76** 21.13** V × T 1 7.52ns 0.28ns Error 12 30.89 4.81

Varieties (V) 1 571.66** 492.61** Treatments (T) 1 4959.95** 1513.98** V × T 1 479.90** 551.16** Error 12 20.36 29.43

Varieties (V) 1 0.31ns 49.55* Treatments (T) 1 633.11** 83.42** V × T 1 43.92ns 1.94ns Error 12 5.74 7.94

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 10: Changes in some grain yield characteristics of control and glasshouse grown maize varieties during winter and summer seasons at maturity

54

2002 under control condition, while glasshouse condition was almost equally detrimental to this

attribute (Fig. 10).

4.1.2 Discussion

In view of the changing environmental condition, mainly related to global warming, the plant

growing patterns are subject to rapid and continuous changes (Porter, 2005). Although maize is a

C4 plant and can withstand relatively higher ambient temperatures (Ashraf and Hafeez, 2004),

growth and yield response of this important cereal crop to glasshouse conditions have not been

comprehensively studies. Maize is a short duration crop and is grown in winter and summer

seasons in Pakistan. It shows differential growth and productivity in these seasons, as different

yields are obtained in both these seasons (Anonymous, 2008). In this study, determination made

for changes in some growth, cob and grain yield attributes of glass canopy (glasshouse) grown

varieties indicated great differences in the selected varieties and treatments. Most importantly,

some interactions of the varieties and treatments for various parameters present in one season

disappeared in the other season at various growth stages. This indicated that prevailing

glasshouse condition modulated the maize growth, although the effects were relatively lesser

evident on the high temperature tolerant variety (Sadaf) than the high temperature sensitive

variety (Agatti-2002) in the glasshouse.

Determination of growth responses at various critical phenological stages indicates the

specific responses of plants under study. This is because during transition from one growth phase

to the other, there is reprogramming of gene expression and sensitivity to changed environmental

conditions may be greater (Milligan et al., 2004; Qin et al., 2004; Wahid and Close, 2007).

These alterations in gene activities result in the developmental changes, as reflected from

changes in plant growth patterns (Srivastava, 2002; Taiz and Zeiger, 2006). In this study, the

determination were made at three phenological stages (seedling, silking and grain filling)

revealed that both the varieties behaved differently at all these growth stages under glasshouse

condition (Figs. 3-8). In addition, the influence of seasons was also well marked, as quite a few

interactions appearing in winter grown plants disappeared in summer grown plants. It is

important to note that at silking stage most of the interaction disappeared in summer. Silking

stage appears to be the most critical for final plant productivity because at this particular stage,

55

number of changes including success of fertilization, seed set and grain filling follow the

reception of pollen by the silk. Similar changes have been reported in maize and other plants

during fertilization (Le Deunff et al., 1993; Wahid et al., 2007).

Determination made for cob (Fig. 9) and grain yield and related (Fig. 10) characteristics

indicated that winter season produced more conspicuous changes than summer season.

Moreover, the effect of glasshouse was also a major factor in producing changes in these

attributes. Data revealed that for cob, most important differences observed across the seasons

were evident in number of grain rows per cob and number of grains per cob (Fig. 9), while for

grain and grain yield components, grain yield per cob, grain yield per plant and harvest index

were more important (Fig. 10). This revealed that glasshouse condition has definitive influence

on the growth and economic yield attributes of maize.

If the differences in the ambient temperature inside and outside the canopy are taken

together, it becomes clear that a rise in the temperature (in the months of May and June) of the

winter sown crop plays a crucial role in the occurrence of changes and producing interaction of

varieties and treatments. Under glasshouse condition in winter grown crop, where the

temperature rises further by 5-7oC and relative humidity declines. On the contrary, in summer

season at silking and grain filling stages there is a continuous decline in temperature and a rise in

relative humidity. These changing climatic conditions appeared to play a role in narrowing down

the differences in the varieties (Fig. 2), thus leading to the disappearance of interactions. In this

context, it is pointed out that a single degree change in ambient temperature is likely to produce a

set of changes in the plants (IPCC, 2007; Wahid et al., 2007).

In conclusion, changes in ambient temperature produce a lot of changes in growth and

yield of maize, and the prevailing glasshouse conditions play a crucial role in this regard across

winter and summer seasons. Investigations on the physiological and biochemical basis of these

changes (as reported in the next sections) will improve out understanding of the underlying

phenomena under the changing growth conditions in the glasshouse.

56

4.2 Leaf pigments and gas exchange properties

4.2.1 Results

a. Chlorophyll a

At seedling stage, in winter season, data revealed non-significant difference in the varieties and

treatments, with no interaction of both factors. In summer season, a non-significant difference in

the varieties and treatments was noted, but a significant interaction of both these factors was

evident for chlorophyll (Chl) a contents. During winter season, in glasshouse grown plants Chl a

contents decreased more in Agatti-2002 as compared to Sadaf. However, in summer season Chl a

contents increased over control in Sadaf but remarkably decreased in Agatti-2002 (Fig. 11).

Chl

orop

hyll

a co

ncen

trat

ion

(mg/

g fr

esh

leaf

tiss

ue)

a aa b

0

2

4

6

8

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0

2

4

6

8

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasonsa aa

b

0

2

4

6

8

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.08ns 0.004ns Treatments (T) 1 0.27ns 1.398ns V × T 1 0.10ns 2.139* Error 12 0.30 0.359

Varieties (V) 1 0.094ns 0.031ns Treatments (T) 1 1.418ns 0.626ns V × T 1 1.090ns 0.022ns Error 12 0.318 0.132

Varieties (V) 1 0.976ns 0.021ns Treatments (T) 1 0.343ns 0.062* V × T 1 1.256* 0.0006ns Error 12 0.23 0.008

Significant at: * P<0.05, ns non-significant Seasons

Fig. 11: Changes in chlorophyll a concentration in the leaves of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

57

At silking stage, in both the seasons, data indicated a non-significant difference in the

varieties and treatments, and there was no significant interaction of both factors for Chl a

contents. In winter and summer seasons, showing no change in control plants, Chl a contents did

not change in Sadaf but decreased in Agatti-2002 under glasshouse condition (Fig. 11).

At grain filling stage, data showed non-significant difference in the varieties and

treatments but there was a significant interaction of both factors in winter season. However, in

summer season, the varieties differed non-significantly, while a significant difference was seen

in the treatments, as well as there was non-significant interaction of varieties and treatments for

Chl a contents. The Chl a contents of winter season plants were greater than those of summer

grown plants. Glasshouse condition increased Chl a contents in Sadaf but decreased in Agatti-

2002. However, in summer season, no specific difference was recorded for changes in Chl a

contents in the varieties under both the conditions (Fig. 11).

b. Chlorophyll b

At seedling stage, statistical results revealed non-significant difference in the varieties and

treatments, as well as there was no significant interaction of both factors in both the seasons for

Chl b. In winter season, in glasshouse grown plants, Chl b contents increased in Sadaf and

reduced in Agatti-2002. However, in summer season the value of this attribute was increased in

Sadaf but did not change in Agatti-2002 under glasshouse condition (Fig. 12).

At silking stage, data analysis revealed non-significant difference in the varieties and

treatments and there was no significant interaction of both factors in both the growing seasons

for Chl b. Overall, the Chl b contents were greater in winter than summer grown plants. At this

stage, glasshouse grown plants of Sadaf displayed increased, while those of Agatti-2002

decreased Chl b contents. However, in summer season, the value of this attribute decreased both

in Sadaf and Agatti-2002 under glasshouse condition (Fig. 12).

At grain filling stage, data exhibited non-significant difference in the varieties but a

significant one in the treatments, while there was no interaction of both these factors in winter

season. However, in summer season, the varieties and treatments differed non-significantly, as

well as there was no interaction of varieties and treatments for Chl b contents. In winter season,

although Chl b contents reduced in both the varieties under under glasshouse condition, Agatti-

2002 was relatively more affected than Sadaf. In the summer season, although Chl b contents

58

were lesser than winter season plants, Sadaf showed increased while Agatti-2002 indicated no

change in Chl b under glasshouse condition (Fig. 12).

c. Total chlorophylls

At seedling stage, statistical treatment of data revealed no significant difference in the varieties,

treatments and no interaction of both factors was notable in both winter and summer season for

total chlorophylls. In winter grown plants, total chlorophylls did not change in Sadaf but

decreased in Agatti-2002 under glasshouse condition. In summer season, the total chlorophylls

increased in Sadaf and decreased in Agatti-2002 (Fig. 13).

Chl

orop

hyll

b co

ncen

trat

ion

(mg/

g fr

esh

leaf

tiss

ue)

0

1

2

3

4

5

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0

1

2

3

4

5

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

0

1

2

3

4

5

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.02ns 0.55ns Treatments (T) 1 0.06ns 0.28ns V × T 1 0.21ns 0.02ns Error 12 0.15 0.13

Varieties (V) 1 0.815ns 0.035ns Treatments (T) 1 0.1060ns 0.374ns V × T 1 0.814ns 0.043ns Error 12 0.454 0.094

Varieties (V) 1 0.453ns 0.749ns Treatments (T) 1 1.353** 0.152ns V × T 1 0.129ns 0.476ns Error 12 0.10 0.168

Significant at: ** P<0.01, ns non-significant Seasons Fig. 12: Changes in chlorophyll b concentration of in the leaves of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

59

At silking stage, in both the seasons, statistical analysis of data indicated no significant

difference in the varieties and treatments and there was no interaction of both factors. Winter

grown plants displayed greater total chlorophyll contents than summer grown plants. In winter

season, total chlorophyll increased in Sadaf but decreased in Agatti-2002 under glasshouse

condition. In summer season, glasshouse condition did not produced remarkable changes in the

total chlorophyll contents of both the varieties (Fig. 13).

At grain filling stage, results revealed significant difference in the varieties and

treatments, with a significant interaction of both factors in winter season. However, in summer

season, the varieties and treatments differed non-significantly and there was no interaction of

varieties and treatments for total chlorophylls. Like previous stage, winter grown plants

Tot

al c

hlor

ophy

ll c

once

ntra

tion

(mg/

g fr

esh

leaf

tiss

ue)

0

3

6

9

12

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0

3

6

9

12

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

a ab

b

0

3

6

9

12

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.01ns 0.65ns Treatments (T) 1 0.57ns 1.03ns V × T 1 0.64ns 2.61ns Error 12 0.68 0.69

Varieties (V) 1 1.462ns 0.132ns Treatments (T) 1 2.298ns 0.032ns V × T 1 3.786ns 0.128ns Error 12 1.006 0.212

Varieties (V) 1 2.758** 0.520ns Treatments (T) 1 3.057** 0.012ns V × T 1 2.189* 0.067ns Error 12 0.29 0.17

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 13: Changes in total chlorophyll concentration of in the leaves of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

60

manifested greater total chlorophyll contents than summer season plants. In winter season, Sadaf

exhibited no changes while Agatti-2002 indicated a decrease in total chlorophyll contents under

glasshouse condition. In summer season, on the other hand, total chlorophyll contents did not

differ much in the control and glasshouse condition in both the varieties (Fig. 13).

d. Chlorophyll a/b ratio

At seedling stage, for summer and winter seasons, results showed no significant difference in the

varieties and treatments and there was no interaction of both factors for this parameter Chl a/b

ratio. The winter grown plants showed a greater Chal a/b ratio than summer grown plants. In

winter season, this ratio was slightly reduced in Sadaf but increased in Agatti-2002. However, in

summer season, Chl a/b ratio reduced in both varieties, although greatly in Agatti-2002 (Fig. 14).

Chl

orop

hyll

a/b

rat

io

0.0

0.5

1.0

1.5

2.0

2.5

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0.0

0.5

1.0

1.5

2.0

2.5

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

0.0

0.5

1.0

1.5

2.0

2.5

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.05ns 0.06ns Treatments (T) 1 1.12ns 0.12ns V × T 1 0.02ns 0.05ns Error 12 0.05 0.02

Varieties (V) 1 0.033ns 0.004ns Treatments (T) 1 0.009ns 0.377* V × T 1 0.000ns 0.005ns Error 12 0.061 0.056

Varieties (V) 1 0.005ns 0.144* Treatments (T) 1 0.176ns 0.068ns V × T 1 0.021ns 0.036ns Error 12 0.050 0.030

Significant at: * P<0.05, ns non-significant Seasons

Fig. 14: Changes in chlorophyll a/b ratio of control in the leaves of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

61

At silking stage, statistical analysis of data revealed non-significant difference in the

varieties, treatments and there was no significant interaction of both factors in winter season.

However, in summer season, the varieties differed non-significantly while a significant

difference was evident in the treatments, as well as no significant interaction of varieties and

treatments for Chl a/b ratio. Winter season plants displayed relatively reduced Chl a/b ratio than

the summer seaon plants. In winter season under glasshouse condition, although this ratio

reduced in both the varieties, but a greater reduction was observed in Agatti-2002. However, in

summer season, this ratio increased in both Sadaf and Agatti-2002, but a greater increase was

noted in the former variety (Fig. 14).

At grain filling stage, in winter season, results revealed non-significant difference in the

varieties and treatments, and there was no significant interaction of both factors. However, in

summer season, the varieties differed significantly while non-significant difference was present

in the treatments, and there was no interaction of varieties and treatments for Chl a/b ratio. In

winter season, although this ratio increased in both Sadaf and Agatti-2002 under glasshouse

condition, a greater increased was evident in Sadaf. In summer season, this ratio was reduced in

both varieties irrespective of the growth condition. Nevertheless, under glasshouse condition, this

ratio decreased more in Sadaf than Agatti-2002 (Fig. 14).

e. Total carotenoids

At seedling stage, in winter season, statistical analysis of results revealed significant difference

among the varieties but non-significant difference in the treatments with a non-significant

interaction of both these factors. However, in summer season, not the varieties but the treatments

indicated significant difference, but no interaction of both these factors was present for total

carotenoids (Car). In winter season, Car increased in Sadaf but decreased in Agatti-2002 under

glasshouse condition. In summer season, although glasshouse condition reduced Car, both the

varieties indicated a similar pattern of change (Fig. 15).

At silking stage, data revealed significant difference in the varieties, treatments and there

was significant interaction of varieties and treatments of both factors in winter season. However,

in summer season, the varieties differed non-significantly while a significant difference was

evident in the treatments, with a significant interaction of varieties and treatments for Car. In

winter season, under glasshouse condition, Sadaf indicated a smaller but Agatti-2002 a greater

62

decrease in Car. In summer season under glasshouse condition, Car increased substantially in

Sadaf but did not change much in Agatti-2002 (Fig. 15).

At grain filling stage, statistical analysis of results more indicated significant difference

in the varieties, while no significant difference was evident in the treatments, as well as there was

a significant interaction of varieties and treatments in winter season. However, in summer

season, the varieties and treatments differed significantly showing a significant interaction of

varieties and treatments for Car. In winter season, glasshouse grown plants of Sadaf indicated

increased Car over control, which decreased in case of Agatti-2002. In summer season, on the

Tot

al c

arot

enoi

ds c

once

ntra

tion

(mg/

g fr

esh

leaf

tiss

ue)

0.0

0.2

0.4

0.6

0.8

1.0

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

a a b ba

b

ab

0.0

0.2

0.4

0.6

0.8

1.0

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasonsab ab

a a

a

ba

a

0.0

0.2

0.4

0.6

0.8

1.0

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.061* 0.0001ns Treatments (T) 1 0.000ns 0.0461** V × T 1 0.006ns 0.0020ns Error 12 0.008 0.004

Varieties (V) 1 0.052** 0.004ns Treatments (T) 1 0.068** 0.009* V × T 1 0.045** 0.016** Error 12 0.001 0.002

Varieties (V) 1 0.059** 0.034** Treatments (T) 1 0.003ns 0.027** V × T 1 0.054** 0.018** Error 12 0.01 0.001

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 15: Changes in total carotenoids concentration of control in the leaves of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

63

contrary, control and glasshouse grown plants of Sadaf indicated similar Car while Agatti-2002

showed reduced Car (Fig. 15).

f. Net rate of photosynthesis

At seedling stage, for winter season, data revealed no significant difference in the varieties, while

significant difference in the treatments and a non-significant interaction of both these factors.

However, in summer season, the varieties differed non-significantly, while significant difference

was evident in treatments, and there was significant interaction of varieties and treatments for net

rate of photosynthesis (Pn). In both the seasons, Pn decreased in both the varieties under

glasshouse condition, although Agatti-2002 was more affected. Of the seasons, glasshouse

condition in winter season was more adverse than in summer season (Fig. 16).

At silking stage, in winter season, analysis of data revealed significant difference in the

N

et r

ate

of p

hoto

synt

hesi

s (µm

ol/m

2 /s)

ba

bc c

0

5

10

15

20

25

Sadaf Agatti 2002 Sadaf Agatti 2002

Seed

ling

ControlGlasshouse

aaa

b

0

5

10

15

20

25

Sadaf Agatti 2002 Sadaf Agatti 2002

Silk

ing

Seasonsa a

a ab

cb

c

0

5

10

15

20

25

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

ling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 4.66ns 0.08ns Treatments (T) 1 91.33** 16.36** V × T 1 0.41ns 8.41** Error 12 1.56 0.84

Varieties (V) 1 49.21** 1.08ns Treatments (T) 1 78.32** 94.28** V × T 1 17.60* 3.65ns Error 12 2.35 3.64

Varieties (V) 1 2.576ns 14.861** Treatments (T) 1 206.928** 102.718** V × T 1 16.040** 14.100** Error 12 0.818 0.717

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 16: Changes in net rate of photosynthesis of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

64

varieties and treatments with a significant interaction of both factors. However, in summer

season, the varieties differed non-significantly while significant difference was evident in the

treatments. Furthermore, there was no interaction of varieties and treatments for Pn. In winter

season, Pn was lesser than that noted in summer season. Although glasshouse condition was

inhibitory to this character in both the varieties, Sadaf performed better than Agatti-2002.

Growing season had a great effect on this attribute of both the varieties (Fig. 16).

At grain filling stage, in winter season, there was a non-significant difference in the

varieties, but a significant difference was observed in the treatments, with a significant

interaction of both factors for Pn. In summer season, however, varieties and treatments exhibited

differences with a significant interaction of both the factors. In winter season, Pn was greater in

Agatti-2002 under control, but substantially decreased under glasshouse condition. In summer

season, both varieties showed similar Pn under control condition, but under glasshouse condition

Sadaf displayed greater Pn than Agatti-2002 (Fig. 16).

g. Transpiration rate

At seedling stage, in both the seasons, data showed non-significant difference in the varieties

and treatments with a non-significant interaction of both the factors for transpiration rate (E). In

winter season, E being similar in both varieties under control, decreased in Agatti-2002 under

glasshouse condition. Contrarily in summer season, this parameter decreased in Sadaf but

increased in Agatti-2002 under glasshouse condition (Fig. 17).

At silking stage, statistical analysis of data showed non-significant difference in the

varieties and treatments with a non-significant interaction of both these factors during winter

season. However, during summer season, there was difference in the varieties and treatments but

a non-significant interaction of both these factors. There was a significant influence of growing

season on this attribute. In winter season, Sadaf and Agatti-2002 showed similar value of E

under both conditions. Contrarily in summer, E was lower in Sadaf under both conditions as

compared to Agatti-2002, but growth condition did not affect E in Sadaf. However, E decreased

remarkably in glasshouse condition during summer in Agatti-2002 (Fig. 17).

At grain filling stage, data revealed non-significant difference in the varieties and

treatments, together with a non-significant interaction of both these factors in winter. However,

in summer season, the varieties and treatments differed significantly with significant interaction

65

of varieties and treatments for E. In winter season, E decreased in Sadaf and increased in Agatti-

2002 in glasshouse. However, in summer although both the varieties showed a decreased E but

this decrease was greater in Agatti-2002 under glasshouse condition (Fig. 17).

h. Water use efficiency

At seedling stage, in winter season, data revealed non-significant difference in the varieties but a

significant one in treatments, together with non-significant interaction of both factors for water

use efficiency (WUE). However, in summer season, the varieties differed non-significantly while

treatments differed significantly, with a significant interaction of varieties and treatments for this

parameter. In winter season, WUE reduced in both the varieties under glasshouse condition, but

Tra

nspi

ratio

n ra

te (m

mol

/m2 /s

)

0

1

2

3

4

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0

1

2

3

4

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

a aab

0

1

2

3

4

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.001ns 0.07ns Treatments (T) 1 0.021ns 0.03ns V × T 1 0.012ns 0.12ns Error 12 0.042 0.05

Varieties (V) 1 0.006ns 4.45** Treatments (T) 1 0.000ns 0.87* V × T 1 0.0004ns 0.08ns Error 12 0.04 0.10

Varieties (V) 1 0.041ns 0.129** Treatments (T) 1 0.002ns 0.145** V × T 1 0.037ns 0.054** Error 12 0.018 0.004

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 17: Changes in transpiration rate of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

66

this decrease was greater in Agatti-2002. In summer, Sadaf showed similar WUE in both

conditions but Agatti-2002 exhibited reduced WUE in glasshouse (Fig. 18).

At silking, data revealed significant difference in the varieties and treatments but there

was no significant interaction of both factors during winter season. However, in summer season,

the varieties differed significantly but non-significant difference was noted in treatments, with no

interaction of these factors for WUE. In winter season, Sadaf indicated greater WUE than Agatti-

2002 under control. However, glasshouse condition reduced WUE in both varieties but greatly in

Agatti-2002. In summer season, again WUE was greater in Sadaf under control condition.

However, glasshouse increased this parameter in Sadaf but markedly reduced in Sadaf (Fig. 18).

Wat

er u

se e

ffic

ienc

y (P

n/E

)

aa

ab

0

2

4

6

8

10

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0

2

4

6

8

10

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

a a a a

b

c

b

c

0

2

4

6

8

10

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.96ns 0.15ns Treatments (T) 1 21.92** 3.85** V × T 1 0.01ns 3.92** Error 12 0.56 0.27

Varieties (V) 1 16.26** 19.78** Treatments (T) 1 33.47** 0.51ns V × T 1 7.44ns 1.98ns Error 12 0.87 0.63

Varieties (V) 1 2.92** 1.80ns Treatments (T) 1 80.27** 39.22** V × T 1 10.51** 5.08* Error 12 0.21 0.74

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 18: Changes in water use efficiency of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

67

At grain filling stage, data revealed significant difference in the varieties and treatments,

with a significant interaction of both factors in winter season. In summer season, on the contrary,

varieties showed non-significant but treatments indicated significant difference with a significant

interaction of varieties and treatments for WUE. Under control condition, WUE was similar in

both the varieties across the seasons. However, glasshouse condition in both the seasons was

damaging to this attribute, although Sadaf performed better than Agatti-2002 in both the seasons

for WUE (Fig. 18).

i. Stomatal conductance

As evident from Fig. 19, at seedling stage, in winter season, data analysis showed no significant

difference in the varieties but a significant one in the treatments, with non-significant interaction

of both the factors. However, in summer season, the varieties and treatments differed

significantly, showing significant interaction of varieties and treatments for stomatal

conductance (gs). In winter season, both the varieties indicated greater gs than those grown in

summer irrespective of the growth condition. Although glasshouse condition reduced this

attribute in both the seasons, Agatti-2002 was more affected than Sadaf.

At silking stage in summer season, data showed significant difference in the varieties but

non-significant difference in the treatments, with a non-significant interaction of both factors for

gs. However, in summer season, there was non-significant difference in the varieties and

treatments with non-significant interaction of both these factors. In winter season, gs was greater

in both the varieties than summer. In winter, Sadaf under glasshouse condition showed greater gs

than controls, which was reduced in Agatti-2002. However, in summer season both the varieties

indicated a reduction in gs, which was proportionately greater in Agatti-2002 (Fig. 19).

At grain filling stage, analysis of data for gs, in winter season, showed significant

difference in the varieties and treatments, but there was no interaction of both factors. However,

in summer season, a significant difference was evident in varieties, a non-significant one in

treatments but a significant interaction of both these factors for gs. At this stage, gs was much

lower in winter than in summer under both the conditions. Glasshouse condition reduced this

attribute more in Agatti-2002 than Sadaf. In summer season, Sadaf showed increased gs over

control in glasshouse condition, which decreased remarkably in Agatti-2002 (Fig. 19).

68

j. Sub-stomatal CO2 concentration

At seedling stage, for winter season plants, statistical analysis of data revealed no significant

difference among the varieties and treatments, and no significant interaction of both these

factors. However, in summer season, the varieties and treatments differed significantly showing

a significant interaction of varieties and treatments for sub-stomatal CO2 concentration (Ci).

Both varieties, showing similar Ci under control condition across the seasons, indicated an

increased Ci under glasshouse condition. However, Agatti-2002 showed a relatively greater

increase than in Ci than Sadaf in both the seasons (Fig. 20).

Stom

atal

con

duct

ance

(mol

/m2 /s

)

ab ab

c

0

0.1

0.2

0.3

0.4

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0

0.1

0.2

0.3

0.4

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasonsa aa

b

0

0.1

0.2

0.3

0.4

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.003ns 0.005** Treatments (T) 1 0.010* 0.016** V × T 1 0.002ns 0.011** Error 12 0.001 0.001

Varieties (V) 1 0.007* 0.001ns Treatments (T) 1 0.000ns 0.003ns V × T 1 0.002ns 0.001ns Error 12 0.000 0.002

Varieties (V) 1 0.005** 0.006* Treatments (T) 1 0.006** 0.004ns V × T 1 0.001ns 0.006* Error 12 0.0003 0.001

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons

Fig. 19: Changes in stomatal conductance of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

69

At silking stage in winter season, data for Ci showed significant difference in the

varieties and treatments, although there was no significant interaction of both factors. However,

in summer season, the varieties and treatments differed non-significantly with no interaction

these factors for this attribute. In winter season, Ci of control plants was lower than those of

summer season plants. Glasshouse condition increased Ci in both the varieties in both seasons,

although such an increase was significantly greater in Agatti-2002 (Fig. 20).

At grain filling stage, in both the seasons, data revealed no significant difference in the

varieties but a significant difference in the treatments, and there was no interaction of both

factors for Ci. In both winter and summer season, Ci was similar in control plants of both the

varieties. Glasshouse condition led to an increase in Ci in both the seasons, although this increase

was relatively greater in Agatti-2002 (Fig. 20).

Subs

tom

atal

CO

2 con

cent

ratio

n (µ

mol

/mol

)

b bba

0

50

100

150

200

250

300

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0

50

100

150

200

250

300

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

0

50

100

150

200

250

300

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 11.90ns 1105.56** Treatments (T) 1 957.90ns 2932.22** V × T 1 123.21ns 1592.01** Error 12 296.20 109.33

Varieties (V) 1 3326.41* 273.90ns Treatments (T) 1 5886.73** 2631.69ns V × T 1 1070.93ns 637.56ns Error 12 425.13 725.98

Varieties (V) 1 852.64ns 368.84ns Treatments (T) 1 5062.32* 7421.82** V × T 1 145.20ns 49.70ns Error 12 549.78 598.44

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 20: Changes in substomatal CO2 concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

70

4.2.2 Discussion

Plant photosynthesis comprises two processes; light reaction and dark reactions. Light reactions

are involved in the generation of reducing powers for the dark reaction. Photosynthetic pigments

(primarily chlorophylls and secondarily carotenoids) are important components of light

harvesting centers in light reactions (Taiz and Zeiger, 2006). Studies show that these

photosynthetic pigments are highly prone to the changes in the environmental conditions

(Pastenes and Horton, 1996a, b), which have been taken as stress sensitivity criteria in wheat

(Ristic et al., 2007, 2008). Quite a few studies report the influences of sub-optimal

environmental conditions on the photosynthetic pigments but those describing the changes in the

photosynthetic pigments in glasshouse grown plant throughout the plant ontogeny are few.

Results of this experiment revealed that both the maize varieties indicated quite a lot

changes in Chl a, Chl b, their total, Chl a/b ratio and Car contents in both the growing seasons

and growth conditions (Figs. 11-15). In general, with remarkable differences in the varieties,

winter grown plants indicated more explicit differences than the summer grown plants at all

phenological stages. Of two chlorophyll species, Chl b was more damaged by prevailing high

temperature condition in the glasshouse (Fig. 12) than Chl a (Fig. 11), leading to an overall loss

of chlorophyll (Fig. 13), thereby causing more yellowing of leaves in Agatti-2002 than Sadaf.

These changes resulted in increased chlorophyll a/b ratio (Fig. 14), which was notably higher in

the winter grown plants. It has been shown that high temperature enhances chlorophyllase

activity that degrades the chlorophylls and reduces their contents (Todorov et al., 2003; Wahid et

al., 2007). From the changes in the chlorophyll concentrations, it can be deduced that sensitivity

of Chl b to glasshouse condition is mainly responsible for the yellowing of leaves, particularly in

winter grown plant. Perusing the prevailing temperature conditions in the canopy grown plants in

winter compared to summer, it can be seen that plants sown in winter months had to face more

adverse temperatures at later growth stages (silking and grain filling), than the summer grown

plants, which do not experience such a high temperature during these growth stages. Thus, it can

be inferred that glasshouse conditions are more detrimental to photosynthetic machinery of the

winter sown plants in the warmers months.

Carotenoids have dual roles in plants. By acting as accessory light harvesting pigments,

they harvest the light and funnel onto the photosystems. The other important role of carotenoids

71

remains the alleviation of oxidative damage on the biological membranes via xanthophylls cycle

(Havaux, 1998). Environmentally stressed tolerant plants, are reported to show greater Car as

compared to respective control plants, which suggest their role in the stress tolerance

(Haldimann, 1997; Wahid and Ghazanfar, 2006; Wahid, 2007). In the present research, it was

noted that tolerant maize variety (Sadaf) under glasshouse condition either showed increased,

steady state or minimal decrease in Car during both the seasons as compared to sensitive variety

(Agatti-2002), which displayed decreased Car contents in both the seasons under glasshouse

condition. However, these changes were more remarkable in the winter than summer season

sown maize plants (Fig. 15). Thus, in line with the previous information (Wahid et al., 2007),

this also substantiated a crucial and profound role of Car in the relatively adverse condition like

glasshouse, where increased temperature is a main determinant of growth.

Plant productivity is assessed on the basis of efficiency of a plant to fix CO2 and

production of photoassimilates by the leaves (source tissue) for export to various sinks for

utilization and storage (Rajcan and Tollenaar, 1999). Maize, like a number of other crop plants,

also shows great changes in CO2 fixation under suboptimal growth conditions (Tollenaar, 1989;

Sinsawat et al., 2004). In this study, the gas exchange properties of maize leaves were studied in

terms of changes in net photosynthetic rate (Pn), transpiration rate (E), water use efficiency

(A/E) stomatal conductance and substomatal CO2 concentration (Ci). Present studies revealed

that growing season and glasshouse condition had a great influence on the gas exchange

attributes of both the varieties. Leaf Pn, E and gs were lowly affected at seedling, reduced more

at silking and reduced the most at grain filling stage under glasshouse condition, whilst Agatti-

2002 showed greater sensitivity to glasshouse condition (Fig. 16, 17 and 19). WUE, derived as a

ratio of Pn and E, indicated an increase in winter grown plants than summer grown plants (Fig.

18), which is contrary to many previous reports. However, Ci indicated a decreased value in

winter than during summer (Fig. 20), which is in contrast to the earlier studies (Ranney and Peet,

1994; Morales et al., 2003). A critical perusal of data indicated that declined in the gas exchange

and CO2 fixation by the maize varieties was mainly due to reduced the conductance of CO2 by

stomata to absorb CO2 and its fixation in the Calvin cycle.

As mentioned above, both photosynthetic pigments and gas exchange parameters are

fundamental processes involved in dry matter yield. Thus, optimal operation of reactions in both

these processes is important. Studies highlighting the proportionate changes in these processes

72

are scanty (Tardy and Havaux, 1999). In this study, it was noted that the pattern of changes in

Chl b was more closely related to that those of gs and Ci. Despite the fact that both systems are

entirely different in nature and composition, these results show that parallel changes in both are

important determinants of maize growth.

In conclusion, despite differences in the growing seasons and varieties glasshouse

conditions were adverse for the photosynthetic systems in maize. Major yardsticks of sensitivity

were loss of chlorophyll and carotenoids in the light reactions, while reductions in the stomatal

conductance and substomatal CO2 concentration in dark reaction of the glasshouse grown maize.

73

4.3 Leaf water, osmotic and membrane characteristics

4.3.1 Results

a. Relative water contents

As presented in Fig. 21, at seedling stage, in winter season, there was non-significant difference

in the varieties and treatments, with a non-significant interaction of both factors. However, in

summer season, the varieties and treatments differed significantly, although there was no

interaction of these factors for relative water contents (RWC). In winter season, the varieties

indicated similar RWC under control, which decreased in more in Agatti-2002 as compared to

Sadaf under glasshouse condition. In summer season, Sadaf displayed greater RWC in both the

conditions, although glasshouse condition reduced this character more in Agatti-2002.

R

elat

ive

wat

er c

onte

nts (

%)

0

20

40

60

80

100

Sadaf Agatti 2002 Sadaf Agatti 2002

Seed

ling

Seasons

ControlGlasshouse

0

20

40

60

80

100

Sadaf Agatti 2002 Sadaf Agatti 2002

Silk

ing

0

20

40

60

80

100

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 42.36ns 565.60** Treatments (T) 1 364.74ns 420.79** V × T 1 63.36ns 79.16ns Error 12 135.42 32.81

Varieties (V) 1 82.96* 552.86** Treatments (T) 1 590.33** 758.55** V × T 1 13.12ns 97.74ns Error 12 14.83 28.91

Varieties (V) 1 71.61* 390.26** Treatments (T) 1 327.34** 225.30** V × T 1 34.69ns 13.65ns Error 12 8.16 15.94

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 21: Changes in leaf relative water contents of control and glasshouse grown maize varieties during winter and summer seasons at seedling and silking stages

74

At silking stage, in both the summer seasons, results revealed significant difference in the

varieties and treatments but there was no significant interaction of both factors for RWC. RWC

was not much different in both the varieties under control condition in both the seasons.

However, glasshouse effect was evident on both the varieties indicated reduction in RWC,

although Agatti-2002 was affected more than Sadaf in both the seasons (Fig. 21).

At grain filling stage too, during both the seasons, data revealed significant difference in

the varieties and treatments with a non-significant interaction of these factors for RWC. At this

stage, RWC was slightly higher in Agatti-2002 than Sadaf in both the seasons. However,

glasshouse condition was almost equally adverse for this attribute in both the varieties (Fig. 21).

b. Leaf water potential

At seedling stage, statistical analysis of data showed no significant difference in the varieties but

a significant difference in the treatments, together with a significant interaction of both the

factors. Contrarily, in summer season the varieties and treatments differed significantly but there

was no interaction of varieties and treatments for leaf water potential. In winter season, the leaf

water potential was similar in Sadaf under both the conditions, but increased under glasshouse

condition in Agatti-2002. Contrarily, in summer season, leaf water potential was increased in

both the varieties in a similar manner under glasshouse condition. Winter grown plants had more

negative leaf water potential than those grown in summer season (Fig. 22).

At silking stage, analysis of data showed no significant difference in the varieties and the

treatments with a non-significant interaction of both the factors during winter season. However,

in summer season, a non-significant difference in the varieties but a significant one in the

treatments was noted, but there was no significant interaction of these factors. In winter season,

the leaf water potential was almost similar in Sadaf and Agatti-2002 under both the conditions.

Contrarily, in summer season, leaf water potential increased in both the varieties in a similar

manner under glasshouse condition as that in the control. Summer grown plants had more

negative leaf water potential than those grown in winter season (Fig. 22).

At grain filling stage, results revealed no significant difference in the varieties and

treatments, and a non-significant interaction of both these factors in both the seasons. In winter

season under glasshouse condition, Sadaf indicated an increase while Agatti-2002 indicated a

decrease in this character under glasshouse condition. However, in summer season Sadaf

75

indicated a decrease while Agatti-2002 an increase in this attribute under glasshouse condition

(Fig. 22).

c. Leaf osmotic potential

At seedling stage, analysis of data showed significant difference in the varieties, treatments

together with more significant interaction of both the factors in winter season. However, in

summer season, the difference in varieties and treatments and their interaction was non-

significant. In winter season, the leaf osmotic potential was similar in Sadaf under both the

conditions, but increased under heat stress in Agatti-2002. Contrarily, in summer season, leaf

osmotic potential decreased in Agatti-2002 but Sadaf showed similar trend in both conditions.

Winter grown plants had more negative leaf osmotic potential than those grown in summer

season (Fig. 23).

Wat

er p

oten

tial (

-MPa

)

b bba

0.0

0.3

0.6

0.9

1.2

1.5

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0

0.3

0.6

0.9

1.2

1.5

Sadaf Agatti 2002 Sadaf Agatti 2002

Silk

ing

0

0.3

0.6

0.9

1.2

1.5

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.011ns 0.025* Treatments (T) 1 0.066** 0.121** V × T 1 0.061** 0.007ns Error 12 0.003 0.003

Varieties (V) 1 0.000ns 0.003ns Treatments (T) 1 0.001ns 0.200** V × T 1 0.000ns 0.005ns Error 12 0.004 0.002

Varieties (V) 1 0.000ns 0.000ns Treatments (T) 1 0.005ns 0.005ns V × T 1 0.016ns 0.016ns Error 12 0.051 0.004

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 22: Changes in leaf water potential of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

76

At silking stage, in both the seasons, data showed non-significant difference in the

varieties, treatments together with non-significant interaction of both these factors. In winter

season, the leaf osmotic potential was better in Sadaf than Agatti-2002 under heat stress

condition. In summer season, leaf osmotic potential decreased a little in both the varieties under

heat stress than control condition (Fig. 23).

At grain filling stage, data revealed non-significant difference in the varieties, but a

significant one in the treatments, although there was no interaction of these factors in the winter

season. However, in summer season there was no significant difference in varieties and

treatments, and no interaction of these factors was evident. In winter season plants, leaf osmotic

potential decreased almost equally both in Sadaf and Agatti-2002 under glasshouse condition.

However, in summer season, under glasshouse condition, Sadaf indicated a decrease while

Agatti-2002 a small increase in this parameter (Fig. 23).

Osm

otic

pot

entia

l (-M

Pa)

b bba

0.0

0.4

0.8

1.2

1.6

2.0

Sadaf Agatti 2002 Sadaf Agatti 2002

Seed

ling

ControlGlasshouse

0.0

0.4

0.8

1.2

1.6

2.0

Sadaf Agatti 2002 Sadaf Agatti 2002

Silk

ing

0.0

0.4

0.8

1.2

1.6

2.0

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.044** 0.0096ns Treatments (T) 1 0.040** 0.0034ns V × T 1 0.044** 0.0001ns Error 12 0.004 0.0060

Varieties (V) 1 0.0038ns 0.0012ns Treatments (T) 1 0.0078ns 0.0042ns V × T 1 0.0153ns 0.0001ns Error 12 0.0312 0.0076

Varieties (V) 1 0.0100ns 0.007ns Treatments (T) 1 0.042* 0.010ns V × T 1 0.002ns 0.026ns Error 12 0.005 0.008

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 23: Changes in leaf osmotic potential of control and glasshouse grown maize varieties during winter and summer seasons at seedling and silking stages

77

d. Leaf turgor potential

At seedling stage, data analysis revealed significant differed in the varieties but non-significant

difference in the treatments, with no interaction of both factors in winter season. In summer

season, on the other hand, the varieties differed non-significantly while the treatments indicated

significant difference with a significant interaction of varieties and treatments. In both winter and

summer, although turgor potential was reduced in both the varieties under glasshouse condition,

Agatti-2002 was affected lesser than Sadaf in winter season but more than Sadaf in summer

season. Growing season had great effect on this attribute in both the varieties (Fig. 24).

At silking stage, statistical analysis of data indicated non-significant difference in the

varieties and treatments with no interaction of both these factors in winter season. However, in

summer season, a non significant difference was noted in the varieties but a significant

Tur

gor

pote

ntia

l (M

Pa)

a a

bc

0

0.3

0.6

0.9

Sadaf Agatti 2002 Sadaf Agatti 2002Se

edlin

g

Seasons

ControlGlasshouse

0

0.3

0.6

0.9

Sadaf Agatti 2002 Sadaf Agatti 2002

Silk

ing

0

0.3

0.6

0.9

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.011* 0.003ns Treatments (T) 1 0.003ns 0.165** V × T 1 0.001ns 0.014* Error 12 0.002 0.002

Varieties (V) 1 0.001ns 0.008ns Treatments (T) 1 0.015ns 0.262** V × T 1 0.017ns 0.006ns Error 12 0.045 0.009

Varieties (V) 1 0.000ns 0.004ns Treatments (T) 1 0.039** 0.030** V × T 1 0.000ns 0.001ns Error 12 0.001 0.002

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 24: Changes in leaf turgor potential of control and glasshouse grown maize varieties during winter and summer seasons at seedling and silking stages

78

difference was evident in the treatments, with non-significant interaction of both these factors.

During winter season, glasshouse condition did not influence this attribute in Sadaf but decreased

in Agatti-2002. In winter season, leaf turgor potential was not affected in Sadaf but reduced in

Agatti-2002. In summer, both the varieties showed a reduction in this attribute (Fig. 24).

At grain filling stage, data revealed non-significant difference in the varieties, but a

significant one in treatments, while there was no interaction of both these factors during summer

and winter seasons. In winter and summer season plants, leaf turgor potential decreased equally

in Sadaf and Agatti-2002 under glasshouse condition (Fig. 24).

e. Hydrogen peroxide concentration

At seedling stage, results revealed significant difference in the varieties and treatments but there

was no interaction of both factors in winter season. However, in summer season, the varieties

differed non-significantly, while the treatments differed significantly, while there was no

interaction of varieties and treatments for this parameter. In winter season, although H2O2

concentration was increased in both the varieties in glasshouse grown plants, Agatti-2002

produced substantially higher H2O2. However, in summer season, the increase in H2O2

concentration in glasshouse grown plants was smaller than winter season plants (Fig. 25).

At silking stage, analysis of data revealed significant difference in the varieties and

treatments with a significant interaction of both the factors in winter season. However, in

summer season, the varieties differed non-significantly, treatments significantly, and there was

no interaction of varieties and treatments for this attributes. In winter season, H2O2 concentration

was greater in Agatti-2002 than Sadaf in glasshouse condition. However, in summer season, both

the varieties showed an increased H2O2 synthesis in both the growth conditions, although,

glasshouse condition was effective in increasing its concentration in both the varieties (Fig. 25).

At grain filling stage, statistical analysis of data revealed significant difference in the

varieties and treatments but there was no interaction of both factors in winter as well as summer

seasons. In winter and summer seasons, Sadaf manifested a lower H2O2 production in both

control and glasshouse grown plants as compared to Agatti-2002. Growing season had a

significant effect on this attribute in both the varieties (Fig. 25).

79

f. MDA concentration

At seedling stage, results showed significant difference in the varieties and treatments with a

significant interaction of both these factors during winter season. However, during summer

season, the varieties and treatments differed significantly while there was no interaction of these

factors for malondialdehyde (MDA) accumulation. Maize plants in both the seasons under

glasshouse condition, indicated an accumulation of MDA, but this tendency was much greater in

the winter season plants. More importantly, Agatti-2002 showed greater MDA accumulation

than Sadaf in winter under glasshouse condition (Fig. 26).

Hyd

roge

n pe

roxi

de c

once

ntra

tion

(µm

ol/g

fres

h tis

sue)

c cb

a

0

15

30

45

60

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

c cb

a

0

15

30

45

60

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

0

15

30

45

60

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 63.14** 2.03ns Treatments (T) 1 277.70** 59.59** V × T 1 21.43ns 5.50ns Error 12 5.39 5.12

Varieties (V) 1 118.58** 14.84ns Treatments (T) 1 328.25** 36.79** V × T 1 71.65** 4.92ns Error 12 1.84 3.92

Varieties (V) 1 112.29** 328.23** Treatments (T) 1 61.68* 15.73* V × T 1 24.73ns 0.47ns Error 12 7.69 3.14

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 25: Changes in hydrogen peroxide concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

80

At silking stage, data showed significant difference in the varieties and treatments

together with a significant interaction of both the factors during both winter and summer seasons.

In winter season, Sadaf did not show the accumulation of MDA, which increased remarkably in

Agatti-2002 under glasshouse condition. Contrarily, in summer season, MDA increased in both

the varieties, but Agatti-2002 indicated markedly greater MDA accumulation than Sadaf under

glasshouse condition (Fig. 26).

At grain filling stage, statistical treatment of data showed non-significant difference in

the varieties, but a significant difference in the treatments together with a significant interaction

of both the factors in winter season. However, in summer season, the varieties and treatments

differed significantly while there was no interaction of varieties and treatments for MDA. In

Mal

ondi

alde

hyde

(MD

A) c

once

ntra

tion

(nm

ol/g

fres

h tis

sue)

c c

b

a

0

5

10

15

20

25

30

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

b b

cc

b

a

b

a

0

5

10

15

20

25

30

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasonsbc cab

a

0

5

10

15

20

25

30

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 15.49** 7.97* Treatments (T) 1 235.28** 6.58* V × T 1 10.83** 1.17ns Error 12 0.95 1.10

Varieties (V) 1 101.46** 48.77** Treatments (T) 1 54.45** 215.90** V × T 1 62.33** 15.87* Error 12 1.84 1.82

Varieties (V) 1 17.45ns 32.60** Treatments (T) 1 178.35** 65.04** V × T 1 45.24* 0.55ns Error 12 6.11 1.56

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 26: Changes in MDA concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

81

winter season, both the varieties accumulated greater MDA than in the summer season. The

prevailing glasshouse condition although increased MDA in both the varieties, but this increase

was greater in Agatti-2002. In summer season too, MDA increased in both the varieties, but was

greater in Agatti-2002 (Fig. 26).

g. Relative membrane permeability

As presented in Fig. 27, at seedling stage, in winter season, statistical analysis of data showed

significant difference in the varieties and treatments, but there was no interaction of both the

factors. However, in summer season, varieties and treatments showed significant difference

Rel

ativ

e m

embr

ane

perm

eabi

lity

(%)

b bba

0

10

20

30

40

50

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

cc

c cb

a

ba

0

10

20

30

40

50

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

c cb

a

0

10

20

30

40

50

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 81.99** 16.03** Treatments (T) 1 209.30** 43.30** V × T 1 5.82ns 21.14** Error 12 8.68 1.44

Varieties (V) 1 428.38** 7.84ns Treatments (T) 1 1783.80** 1065.79** V × T 1 210.41** 44.90* Error 12 6.18 6.10

Varieties (V) 1 32.51** 37.22ns Treatments (T) 1 184.21** 424.44** V × T 1 16.03* 32.093ns Error 12 2.25 13.138

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 27: Changes in relative membrane permeability (RMP) of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

82

together with a significant interaction of both these factors for relative membrane permeability

(RMP). In winter season, the RMP increased in Sadaf as well as in Agatti-2002 under glasshouse

condition, but Agatti-2002 showed higher RMP. Contrarily, in summer season, RMP was

nominally increased in Sadaf but greatly in Agatti-2002 under glasshouse condition (Fig. 27).

At silking stage, analysis of data showed significant differences in the varieties and

treatments with a significant interaction of both the factors in the winter season. However, in

summer season, the varieties differed non-significantly, but the treatments differed significantly

and there was a significant interaction of varieties and treatments for RMP. In both winter and

summer season, the RMP was increased both in Sadaf and in Agatti-2002 under glasshouse

condition, but a substantial increase was observed in the latter variety (Fig. 27).

At grain filling stage, in winter season, data showed significant difference in the varieties

and treatments with a significant interaction of both these factors in the winter season. However,

in summer season, the varieties differed non-significantly, while treatment differed significantly,

although there was non-significant interaction of varieties and treatments for RMP. Winter

grown plants, irrespective of the growth condition, indicated reduced RMP than summer grown

plants. In winter and summer seasons, the RMP was increased in both Sadaf and Agatti-2002

under glasshouse condition. Nonetheless, Agatti-2002 showed relatively higher RMP than Sadaf

in both the seasons (Fig. 27).

h. Leaf free proline

At seedling stage, statistical analysis of results revealed a significant difference in the varieties

and treatments and there was a significant interaction of both factors in winter season. However,

in summer season, the varieties differed significantly, treatments non-significantly, while there

was no interaction of varieties and treatments for this parameter. Leaf free proline accumulation

was greater in winter than summer season, irrespective of the growth condition. Nonetheless, in

winter season, leaf free proline was similar in both the varieties under control but increased more

in Sadaf under glasshouse condition. In summer season, free proline increased in Sadaf but

decreased in Agatti-2002 under glasshouse condition (Fig. 28).

At silking stage, data revealed non-significant difference in the varieties but a significant

difference in the treatments, and there was significant interaction of both factors in winter

season. However, in summer season, the varieties differed significantly while no significant

83

difference in the treatments, as well as there was no interaction of varieties and treatments for

free proline. In winter season, free proline increased in Sadaf, but decreased in Agatti-2002

under glasshouse condition. In summer season, Sadaf showed a substantial increase in free

proline while Agatti-2002 remained at par with its control value (Fig. 28).

At grain filling stage, analysis of data revealed non-significant difference in the varieties,

a significant difference in the treatments, while there was no interaction of both the factors in

winter season. However, in summer season, the varieties differed significantly while no

significant difference was observed in treatments, but there was significant interaction of

varieties and treatments. In winter season, under glasshouse condition although free proline

increased in both the varieties, Agatti-2002 indicated a greater increase than Sadaf. Contrarily in

Free

pro

line (

µmol

/g fr

esh

tissu

e)

c c

c b

a

b

a

d

0

10

20

30

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

cb

a

c

0

10

20

30

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

bcb

a

c

0

10

20

30

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 48.47* 11.56** Treatments (T) 1 198.27** 1.17ns V × T 1 29.83* 25.35** Error 12 5.37 0.27

Varieties (V) 1 3.25ns 233.71* Treatments (T) 1 5.90* 104.16ns V × T 1 33.42** 106.84ns Error 12 1.12 50.34

Varieties (V) 1 74.64ns 20.62* Treatments (T) 1 345.38** 1.99ns V × T 1 61.22ns 68.64** Error 12 25.27 2.25

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 28: Changes in free proline concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

84

summer season, glasshouse condition increased leaf free proline in Sadaf, while reduced it in

Agatti-2002. Growing season had a great effect on this attribute in both the varieties (Fig. 28).

i. Total free amino acids

At seedling stage, in both winter and summer seasons, data showed significant difference in the

varieties and treatments, but with a non-significant interaction of both the factors. In both winter

and summer seasons, the amount of free amino acids accumulation was almost similar in both

the varities, although glasshouse condition caused a substantial increase in their levels in both

varieties (Fig. 29).

Tot

al fr

ee a

min

o ac

ids

(mg/

g fr

esh

wei

ght)

0

30

60

90

120

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0

30

60

90

120

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

c cc c

a

b

a

b

0

30

60

90

120

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 482.00** 138.60** Treatments (T) 1 349.01** 390.96** V × T 1 89.69ns 152.23ns Error 12 21.89 44.53

Varieties (V) 1 1347.73** 245.93** Treatments (T) 1 4492.15** 4290.25** V × T 1 9.43ns 83.46ns Error 12 22.05 31.86

Varieties (V) 1 415.60** 219.31* Treatments (T) 1 1271.57** 1863.88** V × T 1 497.09** 478.42** Error 12 10.81 41.53

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 29: Changes in total free amino acids of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

85

At silking stage, during winter and summer seasons, data showed significant difference in

the varieties and treatments, while there was non-significant interaction of both the factors for

total free amino acids. In winter season, the amount of total free amino acids was lesser in both

the varieties than summer season plants. However, glasshouse condition produced a great

increase in their levels in both the seasons, although this accumulation was greater in Sadaf (Fig.

29).

At grain filling stage, statistical analysis of results showed significant difference in the

varieties, treatments and there was significant interaction of these factors in both winter and

summer season. In both the seasons, free amino acids accumulation was similar in both varieties

under control condition. However, glasshouse condition increased them in both the varieties, but

this increase was substantially greater in Sadaf in both the seasons (Fig. 29).

4.3.2 Discussion

An important consequence of high temperature stress is the evapo-transpirative loss of water

from plant surface, resulting in the dehydration and consequently hampering the cell water status

(Machado and Paulsen, 2001; Mazorra et al., 2002). Under dehydration stress, plants show quite

a lot of metabolic changes; the accumulation of compatible solutes for osmotic adjustment are

the most important one (Zhu, 2003; Farooq et al., 2009). Excessive dehydration from the leaf

surface, due to prevailing high temperature or drought conditions, leads to the disruption of cell

membranes by peroxidation and solublization of membrane lipids (Wen-yue et al., 2001; Jiang

and Huang, 2001; Iba, 2002). Results of these experiments on maize showed that the glasshouse

condition reduced leaf RWC in both the seasons, although varietal difference was evident (Fig.

21). This reduction led to the altered leaf water potential (Fig. 22), osmotic potential (Fig. 23)

and reduced turgor potential (Fig. 24). This revealed that glasshouse conditions were greatly

effective in modulating the plant water relations.

It is important to note that both the varieties behaved differentially in glasshouse

conditions in both the seasons. This appeared to be related to the prevailing environmental

conditions of temperature and humidity in the glass canopies (Fig. 2). In winter grown plants

(sown in February), at the time of harvesting at seedling, silking and grain filling stages, the

temperature sufficiently increased and relative humidity decreased to substantially affect the leaf

86

water status. However, in summer season (sown in August) crop, although the temperature was

rather higher at the sowing time, at the aforesaid critical stages, the temperature inside the

canopy was reduced and relative humidity increased (Fig. 2), which did not influence much the

leaf water relations.

In a number of plant species, there has been an enhanced production of H2O2, which is a

strong reactive oxygen species (Wahid et al., 2007). Although there is the production of H2O2 in

the normally functioning cells (Taiz and Zeiger, 2006), its production beyond limits, particularly

under stress effects leads to the peroxidation of membrane lipids, and as a result enhanced

production of thiobarbituric acid reactive substances (TBARS) is observed; predominant

amongst those is MDA (Wahid et al., 2007). As a result of these changes, there is the loss of

integrity of cellular membranes and enhanced ion-leakage is observed (Yang et al., 1996). In this

study, it was noted that in both the varieties there was a high production of H2O2 under

glasshouse condition in winter grown plants (Fig. 25), which also exhibited the production of

MDA (Fig. 26). These changes consequently led the membranes to become permeable, as

evident from the ion-leakage measured in terms of relative membrane permeability (Fig. 27). All

these changes paralleled well in winter and summer season crops with the prevailing

environmental conditions. Thus, it is convincing that glasshouse conditions during warmer

months of the years are more damaging to the plant functions than in relatively cool months.

Plants exposed to dehydration or osmotic stress conditions show the accumulation of

compatible solutes. The accumulation of free proline and a number of other amino acids has been

shown to play roles in the osmotic adjustment and stabilization of membrane structures under

stressful conditions (Taiz and Zeiger, 2006; Wahid et al., 2007; Ashraf and Foolad, 2007). In

view of the importance of amino acids to the growth of maize (a C4 plant), the determinations

were carried out for free proline alone (Fig. 28) and total free amino acids (Fig. 29). The results

revealed that under glasshouse condition, tolerant maize variety (Sadaf) accumulated a higher

free proline in higher amounts, while there was a general tendency of both the varieties to

accumulate free amino acids under glasshouse (high temperature) condition. A critical view of

the data revealed that in some cases, free proline accumulation constituted 25-40% of the total

free amino acids accumulated, which further substantiates the role of free proline accumulation,

as reported in a number of heat stressed plants (Chiang and Dandekar, 1995; Wahid, 2007;

87

Wahid et al., 2007; Verbruggen and Hermans, 2008). Thus, the accumulation of free proline can

be taken as a reliable criterion of tolerance to glasshouse conditions.

In conclusion, prevailing glasshouse conditions particularly were greatly effective in

hampering the leaf water relation particularly those of winter sown crop. The glasshouse

conditions in winter crop produced oxidative stress on the plants, which was evident from the

increased synthesis of H2O2, MDA and increased permeability to the ion leakage. Greater free

proline accumulation in the tolerant variety not only presented itself as a major amino acid

accumulated in environmental stress tolerance but also indicated it as a reliable criterion of

tolerance to glasshouse condition in maize.

88

4.4 Nutritional relationships

4.4.1 Results

a. Shoot K contents

At seedling stage, in both the seasons, statistical analysis of data revealed significant differences

in the varieties and treatments but there was no significant interaction of both factors for shoot K.

In both the seasons varieties exhibited differential response to accumulation of K in the shoot

tissues. In glasshouse condition during winter season, Sadaf indicated a greater K accumulation

while in winter season Agatti-2002 excelled Sadaf in accumulating this ion (Fig. 30).

Shoo

t K+ (m

g/g

dry

wei

ght)

0

10

20

30

40

50

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

b abab

0

10

20

30

40

50

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

ab aab a

bcc

a b

0

10

20

30

40

50

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 122.03** 343.98** Treatments (T) 1 253.77** 230.27** V × T 1 0.66ns 25.58ns Error 12 10.96 11.86

Varieties (V) 1 9.97ns 3.73ns Treatments (T) 1 0.60ns 3.73ns V × T 1 10.33ns 42.25* Error 12 2.89 4.58

Varieties (V) 1 4.02ns 1.26ns Treatments (T) 1 54.60** 0.13ns V × T 1 10.56* 9.75* Error 12 2.14 2.00

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons

Fig. 30: Changes in shoot potassium concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

89

At silking stage, analysis of results revealed non-significant difference among the

varieties and treatments in both winter and summer season, while there was no significant

interaction of these factors in winter but a significant interaction in summer season. In both

winter and summer season, shoot K was increased in Sadaf and decreased in Agatti-2002 under

glasshouse condition (Fig. 30).

At grain filling stage, statistical analysis of data revealed non-significant difference in the

varieties, while significant difference in the treatments and a significant interaction of both

factors was seen in winter season. However, in summer season, the varieties and treatments

differed non-significantly, but there was significant interaction of varieties and treatments for

shoot K. In winter season, although shoot K was decreased in both varieties under glasshouse

condition, Agatti-2002 underwent a greater reduction. Contrarily, in summer season shoot K

increased in Sadaf and decreased in Agatti-2002 under glasshouse condition (Fig. 30).

b. Shoot Ca contents

At seedling stage, statistical analysis of data revealed non-significant difference in the varieties

while a significant difference in the treatments with a non-significant interaction of both factors

in winter season. However, in summer season, the differences among the varieties, treatments

and their interaction were significant for shoot Ca. In winter season, this attribute was increased

in Sadaf and decreased in Agatti-2002 under glasshouse condition. In summer season, however,

glasshouse condition did not change shoot Ca much in Sadaf but increased under glasshouse

condition (Fig. 31).

At silking stage, analysis of data revealed non-significant difference in the varieties,

treatments and as well as there was no interaction of varieties and treatments for shoot Ca in

winter season. However, in summer season, the varieties differed significantly while non-

significant difference was evident in treatments and there was no interaction of varieties and

treatments for Ca. Comparison of seasons indicated that shoot Ca was higher in winter than in

summer grown plants irrespective of the growth conditions. In winter season, Ca contents

decreased more in Agatti-2002 under glasshouse condition. In summer season, glasshouse

condition increased Ca in Sadaf but did not remarkably change in Agatti-2002 (Fig. 31).

At grain filling stage, statistical analysis of data revealed non-significant difference in the

varieties, treatments, and there was no interaction of varieties and treatments in winter season.

90

However, in summer season, the varieties and treatments differed significantly, but no significant

interaction was evident in them for shoot Ca. In winter season, shoot Ca increased in Sadaf and

decreased in Agatti-2002 under glasshouse condition. However, in summer season, glasshouse

condition increased the shoot Ca more greatly in Sadaf than Agatti-2002. Growth season had an

effect on this attribute (Fig. 31).

c. Shoot Mg contents

At seedling stage, analysis of data revealed no significant difference in the varieties, a significant

difference in the treatments, but there was no interaction of both these factors in winter season.

However, in summer season, the varieties differed significantly while no difference was seen in

the treatments, and there was a significant interaction of varieties and treatments for shoot Mg.

Shoo

t Ca2+

(mg/

g dr

y w

eigh

t)

12.4423076b b12.2692307

b

a

0

5

10

15

20

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0

5

10

15

20

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

0

5

10

15

20

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.48ns 13.21** Treatments (T) 1 10.94* 6.15* V × T 1 8.32ns 7.99* Error 12 2.13 1.01

Varieties (V) 1 4.34ns 4.75** Treatments (T) 1 3.15ns 0.81ns V × T 1 0.77ns 0.15ns Error 12 2.12 0.84

Varieties (V) 1 2.86ns 20.14* Treatments (T) 1 1.27ns 20.14* V × T 1 7.25ns 3.70ns Error 12 1.55 2.18

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 31: Changes in shoot calcium concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

91

At this stage shoot Mg contents were reduced in winter season grown plants as compared to

summer season plants. In winter season, shoot Mg decreased almost equally in Sadaf and Agatti-

2002 under glasshouse condition. In summer season, although Sadaf showed no change but

Agatti-2002 showed a great increase in shoot Mg contents under glasshouse condition (Fig. 32).

At silking stage, data revealed significant difference in the varieties and treatments and

there was significant interaction of both factors in winter season. In summer season, the varieties

and treatments differed non-significantly, with no interaction of varieties and treatments for

shoot Mg. Winter grown plants showed greater shoot Mg content than summer grown plants. In

winter season, shoot Mg decreased in both the varieties, but a greater decrease was noticeable in

Shoo

t Mg2+

(mg/

g dr

y w

eigh

t)

bb

b

a

0

4

8

12

16

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

abb

c

0

4

8

12

16

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasons

0

4

8

12

16

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 1.97ns 28.29** Treatments (T) 1 4.49* 4.75ns V × T 1 0.11ns 6.41* Error 12 0.57 1.17

Varieties (V) 1 17.41** 0.49ns Treatments (T) 1 18.38** 0.18ns V × T 1 3.49** 0.10ns Error 12 0.18 0.10

Varieties (V) 1 5.41* 4.27** Treatments (T) 1 1.08ns 7.67** V × T 1 3.84ns 0.17ns Error 12 0.99 0.30

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 32: Changes in shoot magnesium concentration of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

92

Agatti-2002 under glasshouse condition. In summer season, Sadaf indicated no difference whilst

Agatti-2002 indicated a decrease in shoot Mg contents under glasshouse condition (Fig. 32).

At grain filling stage, data revealed significant difference in the varieties but no

difference was noted in the treatments, while there was no interaction of these factors in winter

season. In summer season, the varieties and treatments differed significantly, while there was no

interaction of both these factors. Shoot Mg was greater in winter than summer season plants. In

winter season, shoot Mg increased in Sadaf while decreased in Agatti-2002 under glasshouse

condition. In summer season, shoot Mg decreased equally in both the varieties (Fig. 32).

d. Shoot phosphate contents

As can be seen in Fig 33, at seedling stage, statistical analysis of results in both the seasons

Shoo

t sol

uble

pho

spha

te (m

g/g

dry

wei

ght)

0

10

20

30

40

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

0

10

20

30

40

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasonsb b

a

b

0

10

20

30

40

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 43.86* 28.78* Treatments (T) 1 39.31* 23.52* V × T 1 0.32ns 0.09ns Error 12 5.71 4.04

Varieties (V) 1 3.03ns 0.35ns Treatments (T) 1 19.25* 11.68** V × T 1 0.082ns 2.73ns Error 12 2.73 1.12

Varieties (V) 1 56.32** 0.68ns Treatments (T) 1 9.20ns 18.01** V × T 1 16.89* 0.75ns Error 12 3.47 1.73

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 33: Changes in shoot soluble phosphate contents of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

93

revealed a significant difference in the varieties and treatments but there was no significant

interaction of both these factors. In winter season, shoot phosphate decreased, while in summer

season, this attribute increased in both varieties.

At silking stage, data analysis revealed non-significant difference in the varieties but a

significant difference in the treatments with a non-significant interaction of these factors in both

the seasons. In both winter and summer season, shoot phosphate increased in both varieties under

glasshouse condition, although this increase was greater in winter grown plants (Fig. 33).

At grain filling stage, data revealed significant difference in the varieties while non-

significant difference in the treatments, and there was a significant interaction of both factors in

winter season. However, in summer season, the varieties differed non-significantly, while the

treatments differed significantly, although there was no interaction of varieties and treatments for

shoot phosphate. In winter season, shoot phosphate increased remarkably in Sadaf but did not

change much in Agatti-2002 under glasshouse condition. In summer season this attribute was

increased almost equally in both the varieties (Fig. 33).

e. Shoot nitrate contents

At seedling stage, in winter season, data analysis revealed significant difference in the varieties,

while non-significant difference in the treatments and with a non-significant interaction of these

factors. However, in summer season, varieties and treatments indicated non-significant

difference, as well as no interaction of both these factors was observed for shoot nitrate. In

winter season, although glasshouse condition increased shoot nitrate in both Sadaf and Agatti-

2002, a greater increase was experienced in the latter variety. In summer season, shoot nitrate

declined in both varieties but this decline was well explicit in Sadaf (Fig. 34).

At silking stage, statistical analysis of data revealed non-significant difference in the

varieties and treatments and there was no significant interaction of both factors in winter season.

However, in summer season, the varieties differed significantly while no difference was evident

in the treatments, but there was a significant interaction of varieties and treatments for shoot

nitrate. In winter, shoot nitrate increased in Sadaf and decreased in Agatti-2002 under glasshouse

condition. In summer season, glasshouse condition decreased this parameter in Sadaf but

increased in Agatti-2002 (Fig. 34).

94

At grain filling stage, statistical analysis of data revealed non-significant difference in the

varieties and treatments, with no significant interaction of both factors in winter season.

However, in summer season the varieties and treatments differed non-significantly, whilst there

was a significant interaction of varieties and treatments for shoot nitrate. In both winter and

summer season, shoot nitrate increased in Agatti-2002 while decreased in Sadaf under

glasshouse condition (Fig. 34).

Shoo

t sol

uble

nitr

ate

(%)

0

0.1

0.2

0.3

0.4

0.5

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Seed

ling

Seasons

ControlGlasshouse

b bb

a

0

0.1

0.2

0.3

0.4

0.5

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Silk

ing

Seasonsab ab

b

a

0

0.1

0.2

0.3

0.4

0.5

Sadaf Agatti 2002 Sadaf Agatti 2002

Winter-2007 Summer-2007

Gra

in fi

lling

Variance sources DF Winter 2007 Summer 2007 Varieties (V) 1 0.0092** 0.016ns Treatments (T) 1 0.0007ns 0.003ns V × T 1 0.0001ns 0.001ns Error 12 0.00002 0.004

Varieties (V) 1 0.117ns 0.008** Treatments (T) 1 0.453ns 0.002ns V × T 1 0.102ns 0.007** Error 12 0.212 0.001

Varieties (V) 1 0.002ns 0.0041ns Treatments (T) 1 0.0001ns 0.0002ns V × T 1 0.001ns 0.010* Error 12 0.0009 0.0015

Significant at: ** P<0.01, * P<0.05, ns non-significant Seasons Fig. 34: Changes in shoot soluble nitrate of control and glasshouse grown maize varieties during winter and summer seasons at seedling, silking and grain filling stages

95

4.4.2 Discussion

Among the multifarious effects of high temperature stress, changes in the mineral nutrition of the

plant have been regarded as the crucial one. It has been shown that maize, millet and many other

plants grown in heat stress environment show great changes in the mineral nutrition including K,

Ca, Mg, N and P (Ashraf and Hafeez, 2004; Wahid et al., 2007). It is important to mention that

most of these studies have been conducted in artificial hot environment. However, studies

conducted under glasshouse environment, which produce high temperature stress along with low

relative humidity are few.

In this part of the manuscript, determinations were made for the changes in major

nutrients including K, Ca, Mg, phosphate and nitrate. All these have been grouped as major

nutrient for plant growth, since they are either the structural or functional constituents (Epstein

and Bloom, 2005). Furthermore, they play multiple roles in physiological and biochemical

phenomena including osmoregulation, action as cofactors in the enzyme activities, metabolites

synthesis and signal transduction (Taiz and Zeiger, 2006). It was noted that, though not too great,

glasshouse condition across the seasons adversely affected the tissue contents of K (Fig. 30), Ca

(Fig.31), Mg (Fig. 32), phosphate (Fig. 33) and nitrate (Fig. 34) in the shoot at three growth

stages. Nonetheless, varietal difference was evident across the seasons; Sadaf appeared to

maintain greater shoot tissue nutrients than Agatti-2002 in most cases. Plants grown in the winter

season indicated more conspicuous changes than those grown in the summer. It is important to

note further that shoot tissue contents of K, Ca and, to some extent, nitrate indicated more clear

trends of changes across the seasons, which suggested a greater role of these nutrients in the

tolerance to glasshouse condition.

As mentioned previously, growth conditions of temperature and relative humidity inside

the glasshouse are more adverse in the summer instead of winter months. In summer months, the

temperature inside the glass canopy was 5-7oC higher while relative humidity was 5-10% lower

than the outside environment (Fig. 2). Heat stress acts as a dehydrative force, and change of

ambient temperature by 1oC can produce considerable changes in the metabolic phenomena

(IPCC, 2007). The root acquisition and shoot transport of the mineral nutrient is greatly

dependent on the prevailing climatic condition, mainly related to evapo-transpirational load on

the plant leaves. In case of glasshouse, where both the temperature and relative humidity are

96

great modulating factors, great changes in the mineral nutrient concentrations in the shoot are

anticipated. These results therefore confirm the assertion that glasshouse conditions are great

growth modulating factors, and plant growth under such conditions is also affected, at least in

part, by the changes in the tissue nutrient concentrations.

In crux, with great varietal difference, changes of temperature and relative humidity

inside the glasshouse across the seasons were mainly responsible for the observed changes in

mineral nutrients. More distinctly, changes in K, Ca and nitrate nutrition were given greater

credence in view of their closer association to the seasonal changes in the environmental

conditions inside the glasshouse.

97

STUDIES AT SENDAI, JAPAN

The studies at Sendai, Japan were carried out to determine the patterns of gene expression in

maize. Although maize is a C4 plant species, it shows reduced growth at supra-optimal

temperatures. The changes in temperature lead to the altered expression of gene. In view of this,

studies were initiated at the Tohoku University, Sendai Japan to determine the changes in the

expression of heat shock protein 70 (hsp70), dehydrin2 (dhn2), stay green gene (sgr2) and

senescence associated gene (sag) in maize at seedling stage in a time course manner (at 1, 3, 6,

254, 48 and 72 h time interval). Results recorded for the time course changes in the growth

attributes and expression of the abovementioned genes are explained below.

98

4.5 Growth and gene expression in maize under heat stress

4.5.1 Results

a. Plant growth attributes

For shoot growth, data indicated no significant difference in the temperature treatments, but a

significant one in the time points with a significant interaction of both these factors. For shoot

fresh and dry weight, temperature treatments and time points indicated significant difference

while there was no interaction of both these factors, while for shoot fresh-to-dry weight ratio

only temperature treatments indicated significant difference (Table 5). As compared to control,

shoot length in the high temperature treated plants indicated a decrease at 1 h time point, was

equal at 3, 6 and 24 h but increased at 48 and 72 h time points. Shoot fresh and dry weight, and

shoot fresh-to-dry weight ratio indicated a consistent decline as compared to controls, although

fresh weight was more sharply decreased at all time points (Fig. 35).

For root growth, data indicated no significant difference in the temperature treatments,

but a significant one in the time points with a non-significant interaction of both these factors.

For shoot fresh and dry weight and shoot fresh-to-dry weight ratio, temperature treatments and

Table 5: Analysis of variance (mean squares) of some growth and expression analysis attributes of maize under control and high temperature stress (42oC) in a time course manner Parameters Temperature

treatment (T) (DF = 1)

Harvest time (H) (DF = 5)

T × H DF = 5

Error DF = 24

Shoot length 0.15ns 2.81** 0.35* 0.09 Shoot fresh weight 625.00** 274.64** 4.07ns 8.50 Shoot dry weight 51.36** 73.03** 0.89ns 3.94 Shoot fresh/dry weight ratio 0.304** 0.034ns 0.004ns 0.019 Root length 1.25ns 3.70** 0.42ns 0.40 Root fresh weight 240.25** 140.69** 9.72ns 5.19 Root dry weight 3.38** 5.44** 0.18ns 0.55 Root fresh/dry weight ratio 28.72** 1.20** 0.40ns 0.17 Dehydrin 2 (dhn2) 0.19ns 0.40** 0.40** 0.07 Heat shock protein 70 (hsp70) 289.57** 120.13** 120.13** 2.76 Senescence associated gene (sag) 35.84** 9.25** 9.25** 0.87 Stay green gene 2 (sgr2) 25.15** 12.21** 12.21** 1.17 Significant at **, P<0.01; *, P<0.05 and ns, non-significant

99

time points indicated significant difference while there was no interaction of these factors (Table

5). Data revealed that shoot length was equal to controls at 1 h time point, decreased at 3 and 6 h,

but increased at 24, 48 and 72 h time points. Root fresh weight and root fresh-to-dry weight ratio

increased while root dry weight decreased at all time points (Fig. 35).

b b ab ab ab a

cb ab ab

a a

0

4

8

12

16

1 3 6 24 48 72

Shoo

t len

gth

(cm

)

Time (h)

ControlHigh temperature

0

10

20

30

40

50

60

70

1 3 6 24 48 72

Shoo

t fre

sh w

eigh

t (m

g/pl

ant)

Time (h)

0

5

10

15

20

25

30

35

1 3 6 24 48 72

Shoo

t dry

wei

ght (

mg/

plan

t)

Time (h)

0.0

0.5

1.0

1.5

2.0

2.5

1 3 6 24 48 72

Shoo

t fre

sh/d

ry w

eigh

t ra

tio

Time (h)

0

3

6

9

1 3 6 24 48 72R

oot l

engt

h (c

m)

Time (h)

ControlHigh temperature

0

10

20

30

40

1 3 6 24 48 72

Roo

t fre

sh w

eigh

t (m

g/pl

ant)

Time (h)

0

3

6

9

1 3 6 24 48 72

Roo

t dry

wei

ght

(mg/

plan

t)

Time (h)

0

2

4

6

8

1 3 6 24 48 72

Roo

t fre

sh/d

ry w

eigh

t rat

io

Time (h)

Fig. 35: Time course changes in some growth attributes of maize seedlings grown under control (27oC) and heat stress (42oC) conditions

100

b. Expression analysis of genes

Data recorded on the transcriptional analysis of dhn2 gene showed that time points, not the

temperature treatments, indicated a significant difference with a significant interaction of both

these factors. However, for hsp70, sag and sgr2 genes, the differences in temperature treatments

and time points, and their interactions were significant (Table 5). As evident from Fig. 36, there

was a strong expression of all the four genes in the heat treated maize shoots. The transcriptional

level of these genes (expressed as relative ratio to respective controls) was much higher during

early hours of exposure to high temperature stress, but declined later (Fig. 36). However, at 48

and 72 h of exposure to high temperature stress transcription of dhn2 followed by sag again

began to increase, while levels of all these genes remained unaffected under high temperature

stress (Fig. 36).

4.5.2 Discussion

This study was aimed at to determine the gene expression in maize under high temperature

stress. It is widely accepted that environmental stress tolerance is a multigenic phenomenon, and

involves the up- or down-regulation of a number of genes (Bohnert et al., 2006; Walia et al.,

2006; Tomassami et al., 2008). Most important genes involved in high temperature tolerance are

HSPs (Schöffl et al., 1999; Kotak et al., 2007) while other genes of importance are late

embryogenesis abundant (LEA), specifically the dehydrin genes (Porat et al., 2004; Svensson et

al., 2002; Wahid and Close, 2007).

This study reporting the short term (hourly) response of maize indicated differential

growth responses of shoot and root to high temperature. Although shoot and root length were not

influenced by heat stress over such a short period of time, there was a consistent loss in fresh and

dry weight of both shoot and root upon exposure to heat stress (Fig. 35). This response appeared

due to the evapo-transpirational loss of water, since heat stress acts as a dehydrative force

(Wahid et al., 2007). This loss of water is a major factor in affecting the metabolic phenomena,

while the changes in intrinsic metabolite levels immediately signal the expression of pertinent

genes over a short period of time to cope with the stress effects (Bohnert et al., 2006; Buchanan-

Wollaston et al., 2005; Wahid and Close, 2007).

101

In view of the reported varietal differences in various crop plants for responses to these

stresses (Cramer and Schmidt, 1995; Claudio et al., 2006; Wahid et al., 2007), here the

determinations were made on the time course changes in the transcriptional levels of four genes;

senescence associated gene (sag), stay green2 gene (sgr2), heat shock protein gene 70 (hsp70)

and dehydrin gene 2 (dhn2). A high level expression of the transcriptional levels all these genes

at 1 h after exposure to high temperature stress (Fig. 36) indicated the possibility of elicitation of

Fig. 36: Gene expression in maize under heat stress: Agarose gel showing the molecular weight of the senescence associated gene (sag), stay green gene (sgr2), heat shock protein 70 (hsp70) gene and dehydrin 2 (dhn2) gene. Bar charts show the transcriptional levels of sag, sgr2, hsp70 and dhn2 genes, respectively studies in a time course manner

Elec

troph

retic

exp

ress

ion

of g

enes

b b b b b b

a

b bb

ab

b

0

2

4

6

8

10

12

1 3 6 24 48 72

sag

Time (h)

ControlHigh temperaturePoly. (High temperature)

b b b b b b

a

abb b

bb

02468

101214

1 3 6 24 48 72

srg2

Time (h)

b b b b b b

aab

b b b b

-505

1015202530

1 3 6 24 48 72

hsp7

0

Time (h)

ab ab ab ab ab ab

a

bb b

ab ab

0.0

0.5

1.0

1.5

2.0

2.5

dhn2

Tran

scrip

tion

leve

ls o

f gen

es (r

elat

ive

ratio

to re

spec

tive

cont

rols

)

102

stress response triggered by certain signals under heat stress (Bohnert et al., 2006; Wahid et al.,

2007). In view of the fact that heat stress has multiple effects on the plant growth and

development, the expression of sag and dhn2 is a direct high temperature stress response. Heat

stress, among the others, has two major effects; senescence and dehydration of living tissues (Liu

and Huang, 2000; Spano et al., 2003). However, induction of sgr2 a stay green response gene

(Harris et al., 2007) and hsp70 (Wahid et al., 2007) appear to be a protective strategy under high

temperature. It is important to notice that both sag and dhn2 again showed an increase in the

transcriptional level, while sgr2 and hsp70 failed to accomplish this tendency. This was further

evident from a negative correlation of transcriptional level of hsp70 with shoot length (r = -

0.871; P<0.05), root length (r = -0.833; P<0.05) and shoot dry weight (r = -0.893; P<0.05) and

that of sgr2 with shoot length (r = -0.919; P<0.01). This revealed that sensitivity of maize to high

temperature stress can be, at least, partly assigned to failure to express hsp70 and sgr2 under

prolonged high temperature spell.

Studies show the coexpression of various genes, as stronger response to prevailing stress

conditions (Weston et al., 2008). The expression of four genes in this study was more or less

similar, which indicated the possibility of their coexpression. To substantiate this possibility,

regression analysis and correlation coefficient of all the genes was carried out. It was noted that

dhn2 was strongly regressed with sag, while hsp70 and sag were only weakly regressed with

sgr2 gene (Fig. 37). This finding further confirmed the assertion that both dhn2 and sag were

coexpressed to display the high temperature sensitivity response in maize.

y = 9.26x - 1.23r = 0.533ns

05

10152025

0.0 1.0 2.0

hsp7

0

dhn2

y = 4.50x - 0.84r = 0.934**

02468

0.0 1.0 2.0

sag

dhn2

y = 4.30x - 0.99r = 0.776ns

02468

10

0.0 1.0 2.0

srg2

dhn2

y = 0.15x + 2.01r = 0.533ns

02468

0 20 40

sag

hsp70

y = 0.28x + 0.81r = 0.874*

02468

10

0 10 20 30

sgr2

hsp70

y = 0.94x - 0.15r = 0.820*

02468

10

0 5 10

srg2

sag Fig. 37: Trendlines and interrelationships of transcriptional levels of four genes under high temperature stress in maize at seedling stage (Significant at **, P<0.01; *, P<0.05 and ns, non-significant)

103

In summary, maize seedlings showed sensitivity to high temperature stress, which was

evident in terms of morphological (reduction in shoot fresh weight, dry weight of shoot and root

and a reduction in fresh-to-dry weight ratio) and molecular responses. The molecular studies

suggested that the maize sensitivity to high temperature was mainly due to enhanced

coexpression of sag and dhn2 and failure to express hsp70 and sgr2 during relatively long term

exposure to heat stress.

104

GENERAL DISCUSSION

Stress has the potential to produce injury, which occurs as a result of aberrant metabolism and

leading to reduction in growth, yield or even death of the plant or plant parts. Plants undergo a

depression in visual growth and development, but the extent of reduction depends greatly upon

the type of stress, its severity and duration (Zhu, 2003; Taiz and Zeiger, 2006). Greater dry

weight results from the available photosynthetic area together with enhanced capacity of leaves

to photosynthesize (Karim et al., 2000; Huve et al., 2006; Suárez and Medina, 2006). The

observed changes in the maize varieties across the seasons in growth attribute in this study

indicate the possibility of greater effects of heat in restricting the photosynthetic capacity of

maize in dry matter production at various growth stages.

In this study, the determination made at three phenological stages (seedling, silking and

grain filling) showed differential behavior of both the varieties at all growth stages under

glasshouse condition. Appearance/disappearance of quite a few interactions of the parameters

indicated the influences of seasonal changes on these attributes. In this respect, silking stage was

the most important, where most of the interaction appearing during winter season disappeared

during summer. Silking stage is more critical for final plant productivity because at this

particular stage, number of changes including success of fertilization, seed set and grain filling

follow the reception of pollen by the silk (Le Deunff et al., 1993), which ultimately determine

the final plant productivity. These data substantiated that the effect of glasshouse to be major

determinant of changes in various attributes.

It has been established that both light and dark reactions of photosynthesis are prone to

adversaries of environmental stresses (Wahid and Rasul, 2005). Photosynthetic pigments have a

considerable significant, as any reduction/loss in their content is likely to substantially influence

the dry matter production (Wahid, 2007). Likewise, enhanced gaseous exchange capacity of

plants is a real determinant of productivity under stressful conditions (Lawlor and Cornic, 2002).

Among the various gas exchange parameters, net rate of photosynthesis, transpiration rate,

photosynthetic water use efficiency, stomatal conductance and substomatal CO2 concentration

are of great significance in the exhibition of dry matter and grain yields under stress (Morales et

al., 2003; Omami and Hammes 2006). In the present study, although both varieties showed a

reduction in chlorophyll, a, b and their total, and carotenoids, Sadaf showed either maintained or

105

increased amounts of chlorophyll and carotenoids content under glasshouse conditions as

compared to Agatti-2002. Among the gas exchange attributes, net rate of photosynthesis,

transpiration rate and substomatal CO2 concentrations were of greater importance in tolerance to

glasshouse condition. Tardy and Havaux (1999) showed that chlorophyll loss in Syrian barley

had no relationship with loss of the photosynthetic activity of leaves. From this study it emerges

that tolerance to glasshouse conditions is strongly associated with increased content of

photosynthetic pigments. Increased content of carotenoids is of greater significance, since they

have dual role in the plant leaves; light harvesting and stabilization of photosynthetic membranes

(Havaux, 1998; Rmiki et al., 1999; Wahid et al., 2007).

Leaf water relations are hampered under any type of stress condition, which has a great

influence on the dry matter production by the plants (Machado and Paulsen, 2001; Wahid and

Close, 2007). In this research, both varieties behaved differentially to glasshouse conditions

across the seasons, mainly because of prevailing environmental conditions of temperature and

humidity in the glass canopies. The glasshouse conditions greatly hampered the leaf water,

osmotic and turgor potentials. Leaf osmotic potential on the other hand was much more reduced

under glasshouse condition, which displayed remarkable changes in the leaf turgor. Response of

Sadaf under glasshouse condition was better as revealed from higher RWC and maintenance of

greater turgor under glasshouse condition.

Apart from other effects, there is a consensus over one common facet of heat stress

regarding the loss of membrane integrity making them more permeable due to peroxidation of

lipid component, reduced production of antioxidants and a concomitant enhanced production of

reactive oxygen species (Blum and Ebercon, 1981; Yang et al., 1996; Hernandez et al., 2000;

Rizhsky et al., 2002; Rahman et al., 2004; Rachel and Dolan, 2006). Amongst various reactive

oxygen species (ROS), hydrogen peroxide (H2O2) is the most stable and long-lived molecule,

which damages the cellular membranes (Dat et al., 2000; Uchida et al., 2002; Steven et al.,

2003). Enhanced membrane stability and reduced generation of ROS are taken as important

criteria of stress tolerance (Wahid et al., 2007). In the present investigation although varietal

differences were noted for increased relative membrane permeability, RMP (a measure of

membrane stability) and H2O2 content, the varieties with reduced RMP and H2O2 content

displayed better tolerance to the prevailing glasshouse condition. This revealed that stress

tolerance in maize is associated to the improved stabilization of membranes in addition to other

106

factors. It is further pointed out that accumulation of free proline, as noted here comprised about

30-40% of the total free amino acids. This accumulation appeared to play pivotal role in

producing tolerance of winter crop to glasshouse condition. The roles played by free proline

accumulation appear to be related to the osmotic adjustments and stabilization of membrane

structure (Wahid et al., 2007; Verbruggen and Hermans, 2008).

Plant mineral nutrition is another important aspect of plant growth and development

which is greatly influenced by the prevailing conditions, especially those of temperature and

relative humidity (Epstein and Bloom, 2005; Farooq et al., 2009). Many studies conducted under

artificial heat stress showed great changes in the mineral nutrient contents in different plants

(Epstein and Bloom, 2005; Ashraf and Hafeez, 2004; Wahid et al., 2007). However, studies

reporting the changes in plant mineral nutrition under glasshouse conditions are scarce. Here

experiments conducted on five major nutrients (K, Ca, Mg, phosphate and nitrate) showed that,

though not too great, glasshouse condition across the seasons adversely affected the tissue

contents of all these nutrients in the shoot at three growth stages. However, varietal difference

was evident across the seasons; Sadaf maintained greater shoot tissue nutrients than Agatti-2002

in most instances. Winter grown plants indicated more conspicuous changes than those in grown

the summer. Among the nutrients, K, Ca and nitrate indicated more clear trends of changes

across the seasons, suggesting their greater role in the tolerance to glasshouse conditions.

It has been established that stress tolerance is a multigenic phenomenon, involving the

up- or down-regulation of a number of genes (Bohnert et al., 2006; Walia et al., 2006;

Tomassami et al., 2008). This study reporting the short term (hourly) response of maize

indicated differential growth responses of shoot and root to high temperature, primarily related to

the evapo-transpirational loss of water (Wahid et al., 2007). These quantitative changes are the

outcome of modulated activities of various genes. Thus the determinations were made on the

time course changes in the transcriptional levels of four stress-related genes; senescence

associated gene (sag), stay green2 gene (sgr2), heat shock protein gene 70 (hsp70) and dehydrin

gene 2 (dhn2). A high level expression of the transcriptional levels of all these genes at 1 h after

exposure to high temperature stress indicated the possibility of elicitation of stress response

triggered by certain signals under heat stress (Bohnert et al., 2006; Wahid et al., 2007). Among

these genes, the coexpression of sag and dhn2 appears to be a direct high temperature stress

107

response, since senescence and dehydration of living tissues are the direct consequences of heat

stress response (Liu and Huang, 2000; Spano et al., 2003).

In essence, maize is generally sensitive to environmental stresses and these affects are

evident on the photosynthetic pigments, gas exchange and water relations. With great varietal

difference, changes of temperature and relative humidity inside the glasshouse across the seasons

were mainly responsible for the observed changes in mineral nutrients. More distinctly, changes

in K, Ca and nitrate nutrition were given greater credence in view of their closer association to

the seasonal changes in the environmental conditions inside the glasshouse. At molecular level,

coexpression of dhn2 and sag determined the heat sensitivity response in maize. Future research

aimed at large scale gene expression and crop modeling for growing maize in glasshouse

conditions are likely to increase our understanding for growing maize in the upcoming climatic

changes.

108

FUTURE PROSPECTS

It is well accepted that greenhouse effect is changing the crop growth patterns throughout the

globe. Thus is a firm need to carry out concerted efforts to get requisite crop productivity from

the available resources under changing climatic conditions. These studies provided baseline

information on the growth and physiological responses of maize, a C4 plant species, to

glasshouse conditions. Further studies on the metabolic changes and gene expression patterns of

the glasshouse grown plants at various critical growth stages and modeling the crop productivity

on would be imperative.

109

CHAPTER-5 SUMMARY

Environmental stresses are serious threats to agricultural productivity around the globe. The

increasing menace of environmental stresses necessitates finding strategies to cope with their

ever-increasing adversaries. In view of the changing climatic conditions mainly related to

greenhouse effect, this study was focused on determining the responses of two differentially heat

tolerant maize varieties to glasshouse condition. The parameters studied included growth, water

relations, gas exchange, photosynthetic pigments, oxidative damage and gene expression.

Results revealed that changes in ambient temperature produce an array of changes in the

growth and yield of maize, and the prevailing glasshouse conditions play a crucial role in this

regard across winter and summer seasons. Investigations on the physiological and biochemical

basis of these changes (as reported in the next sections) will improve our understanding of the

underlying phenomena. Despite differences in the growing seasons and varieties glasshouse

conditions were adverse effects on the photosynthetic systems in maize. Major indicators of

sensitivity were loss of chlorophylls and carotenoids in the light reactions, while reductions in

the net photosynthesis and stomatal conductance in the dark reaction of the glasshouse grown

maize leaves. Prevailing glasshouse conditions particularly were greatly effective in hampering

the leaf water relation particularly those of winter sown crop. The glasshouse conditions in

winter crop produced oxidative stress on the plants, which was evident from the increased

synthesis of H2O2, MDA and increased permeability to the ion leakage. Greater free proline

accumulation in the tolerant variety not only presented itself as a major amino acid accumulated

in environmental stress tolerance but also indicated it as a reliable criterion of tolerance to

glasshouse condition in maize. With great varietal difference, changes of temperature and

relative humidity inside the glasshouse across the seasons were mainly responsible for the

observed changes in mineral nutrients. More distinctly, changes in K, Ca and nitrate nutrition

were given greater credence in view of their closer association to the seasonal changes in the

environmental conditions inside the glasshouse. Maize seedlings showed sensitivity to high

temperature stress, which was evident in terms of reduction in fresh and dry weight of shoot and

root and a reduction in fresh-to-dry weight ratio, and molecular responses. The molecular studies

110

suggested that the maize sensitivity to high temperature was mainly due to enhanced expression

of sag and dhn2 and failure to express hsp70 and sgr2 during relatively long term exposure.

In crux, although the varieties showed decreasing trend in growth and physiological

attributes response of Sadaf was better to heat stress. These studies indicated that changes in

physiological attributes are genetically related in maize, which provide a great room for

improvement of maize for enhanced net rate of photosynthesis, water use efficiency,

photosynthetic pigments reduced membrane permeability and production of reactive oxygen

species. Selection of varieties showing enhanced expression of heat shock protein and stay green

genes could be a promising approach for successfully growing maize in the upcoming

environmental conditions, when high temperature and low humidity could be likely threats to

maize production.

111

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